Method for screening compounds to determine those which enhance islet cell activity and/or survival and uses therefor

ABSTRACT

In accordance with the present invention, it has been discovered that glucose and incretin hormones promote pancreatic islet cell survival via the calcium and cAMP dependent induction, respectively, of the transcription factor CREB. Specifically, a signaling module has been identified which mediates cooperative effects of calcium and cAMP on islet cell gene expression by stimulating the dephosphorylation and nuclear entry of TORC2, a cytoplasmic CREB coactivator. These findings provide a novel mechanism by which CREB activates cellular gene expression, depending on nutrient and energy status, and facilitate development of assays to identify compounds which modulate the role of TORCs.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 11/398,477, filed Apr.4, 2006, now issued as U.S. Pat. No. 7,485,434, which in turn claimspriority from U.S. Patent Application No. 60/668,407, filed Apr. 4,2005, now expired, the entire contents of each of which is herebyincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to methods for screening compounds todetermine if such compounds are capable of enhancing islet cell activityand/or survival, capable of promoting CREB-mediated gene expression inislet cells, capable of effecting transport of Transducers of RegulatedCREB (TORCs) from the cytoplasm into the nucleus of an islet cell,capable of effecting interactions between TORCs and member(s) of the14-3-3 family of proteins, and the like. In additional aspects, thepresent invention relates to methods for enhancing islet cell activityand/or survival.

BACKGROUND OF THE INVENTION

Under feeding conditions, elevations in blood glucose and circulatingincretin hormones such as glucagon like peptide-1 (GLP-1) stimulateislet survival in part via the activation of the transcription factorCREB (Jhala et al., 2003). Elevations in glucose stimulate insulinsecretion and islet cell gene expression via closure of K_(ATP) channelsand subsequent influx of calcium through activated L-type calciumchannels (Newgard and McGarry, 1995). In contrast, GLP-1 has been foundto promote islet cell survival and proliferation by activation of thecAMP pathway (Hui et al., 2003). Transgenic mice expressing a dominantnegative CREB polypeptide in islets develop diabetes with apoptosis ofinsulin producing beta cells due in part to reduced expression of IRS2,a direct target of CREB activity (Jhala et al., 2003).

cAMP promotes the expression of cellular genes by triggering the PKAmediated phosphorylation of CREB at Ser133 (Gonzalez and Montminy,1989). Phosphorylation of CREB at Ser133 in turn stimulates target geneexpression by enhancing recruitment of the histone acetylase coactivatorparalogs CBP and P300 (Arias et al., 1994; Chrivia et al., 1993; Kwok etal., 1994). The structure of the CREB:CBP complex, using relevantinteraction domains, called KID and KIX, respectively, reveals thatphospho (Ser133) forms direct contacts with residues in KIX that accountfor half of the free energy of complex formation (Parker et al., 1998;Radhakrishnan et al., 1997). Binding of KID to KIX also promotes arandom coil to helix transition in KID that favors formation ofhydrophobic contacts with residues lining a shallow groove in KIX.

In addition to cAMP, CREB is Ser133 phosphorylated in response to anumber of stimuli, including growth factors, shear stress, and UV light(Mayr and Montminy, 2001). A number of these stimuli, however, areincapable of promoting target gene activation via CREB per se due inpart to secondary phosphorylation of CREB at inhibitory sites. Neuronaldepolarization triggers phosphorylation of CREB not only at Ser133 butalso at Ser142 and Ser143 (Kornhauser et al., 2002), for example, andthese modifications destabilize the CREB:CBP complex by electrostaticrepulsion (Kornhauser et al., 2002; Parker et al., 1998).

The ability of calcium signals to promote CREB dependent transcriptionwhile apparently blocking CBP recruitment, at least via the KID domain,is indicative of the potential presence of other coactivators thateither mitigate these effects or function independently of CBP/P300. Theinvolvement of a distinct CREB coactivator in promoting calciumdependent gene expression is further indicated by studies in whichaddition of calcineurin antagonists are observed to blockcalcium-stimulated CREB activity without affecting levels of CREB Ser133phosphorylation (Schwaninger et al., 1995). The identification of suchputative coactivator(s), however, remains elusive.

Although the KID domain in CREB is thought to mediate target geneactivation in response to most extracellular stimuli, other regions,most notably the bZIP DNA binding/dimerization domain, have also beenimplicated in this process. In previous studies using GAL4-CREB fusionproteins to define domain requirements for transcriptional activation,for example, both KID and bZIP domains were found to contributeimportantly to cAMP and KCl responsiveness (Bonni et al., 1995a; Shenget al., 1991). These results are also indicative of the involvement ofadditional cofactors that promote cAMP and calcium dependenttranscription through an interaction with the CREB bZIP domain.Consistent with the ability of this region to recruit components of thetranscriptional apparatus, the CREB bZIP domain has been found to act asa potent repressor of numerous transcription factors when over-expressedin various cells (Lemaigre et al., 1993).

Accordingly, there is a need in the art for methods to identifycompounds that modulate the above-described interactions. Such compoundswill find use in a variety of applications, such as, for example,enhancing islet cell activity and/or survival, promoting CREB-mediatedgene expression in islet cells, effecting transport of Transducers ofRegulated CREB (TORCs) from the cytoplasm into the nucleus of isletcells, effecting interactions between TORCs and member(s) of the 14-3-3family of proteins, and the like.

SUMMARY OF THE INVENTION

In accordance with the present invention, it has been discovered thatglucose and incretin hormones promote pancreatic islet cell survival viathe calcium and cAMP dependent induction, respectively, of thetranscription factor CREB. Specifically, a signaling module has beenidentified which mediates cooperative effects of calcium and cAMP onislet cell gene expression by stimulating the dephosphorylation andnuclear entry of TORC2, a cytoplasmic CREB coactivator. The modulecomprises a cAMP regulated sucrose-nonfermenting-1 (snf1)-like proteinkinase called SIK2 (and related family members) and the calciumregulated phosphatase calcineurin, both of which associate with TORC2 inthe cytoplasm. TORC2 is repressed under basal conditions through aphosphorylation dependent interaction with 14-3-3 proteins. cAMP andcalcium signals stimulate CREB target gene expression via complementaryeffects on TORC2 dephosphorylation; cAMP disrupts the TORC2-associatedactivity of SIK2 and related family members, whereas calcium inducesTORC2 dephosphorylation via calcineurin. The results described hereinestablish that glucose and incretin hormones exert synergistic effectson CREB activity and islet cell survival by targeting a signaling modulethat contains TORC2-associated kinase and phosphatase activities,respectively.

In recent high-throughput expression screens to identify novelmodulators of CREB activity, a family of CREB coactivators, referred toas Transducers of Regulated CREB activity (TORCs) have beencharacterized (see, for example, Conkright et al., 2003a; and Iourgenkoet al., 2003). The three exemplary TORC family members identified thusfar share a highly conserved N-terminal coiled-coil domain that mediatesa direct association with the bZIP domain of CREB. The presentdisclosure establishes that TORC2 is a cytoplasmic co-factor thattranslocates to the nucleus in response to cAMP and calcium signalswhere it modulates CREB target gene expression. TORC2 shuttling activityis regulated by associated protein kinase (e.g., SIK2 or related familymembers) and phosphatase (e.g., calcineurin) activities that modulatelevels of TORC phosphorylation. These findings provide a novel mechanismby which CREB activates cellular gene expression, depending on nutrientand energy status, and facilitate development of assays to identifycompounds which modulate the role of TORCs.

Mammals achieve energy balance by modulating hepatic glucose outputdepending on nutritional status (Saltiel and Kahn, 2001). Elevations inglucagon during fasting trigger the gluconeogenic program, for example,via the cAMP responsive factor CREB (Herzig et al., 2001). By contrast,exercise and other stressors that deplete cellular ATP levels inhibitgluconeogenesis via the AMP kinase pathway, although the underlyingmechanism has remained elusive (Kahn et al., 2005). In accordance withthe present invention, it has been discovered that fasting andenergy-sensing pathways regulate the gluconeogenic program in liver bymodulating the nuclear entry of a transcriptional coactivator calledTransducer of Regulated CREB Activity 2 (TORC2) (see, for example,Conkright et al., 2003a; Iourgenko et al., 2003; Screaton et al., 2004;and Bittinger et al., 2004). Thus, under feeding conditions, TORC2 issequestered in the cytoplasm via phosphorylation at Ser171 (see Screatonet al., 2004).

Glucagon administration promotes rapid Ser171 dephosphorylation, nucleartranslocation, and recruitment of TORC2 to gluconeogenic promoters inliver. Hepatic TORC2 over-expression induces fasting hyperglycemia,whereas knockdown of TORC2 leads to fasting hypoglycemia and silencingof the gluconeogenic program. Following prolonged exposure to glucagon,TORC2 activity is attenuated by a negative feedback loop involving theCREB mediated induction of SIK1, a Ser/Thr kinase that phosphorylatesTORC2 at Ser171 (Screaton et al., 2004; and Katoh et al., 2004).Knockdown of SIK1 enhances TORC2 activity on gluconeogenic genes,whereas SIK1 over-expression silences the gluconeogenic program andpromotes fasting hypoglycemia in mice. Similarly, induction of the AMPK(AMP-activated protein kinase) pathway with an AMP analog inhibits TORC2activity on gluconeogenic genes; these effects are rescued by expressionof phosphorylation-defective Ser171Ala TORC2. Since a majority ofindividuals with Type II diabetes exhibit fasting hyperglycemia due toelevated hepatic gluconeogenesis, compounds that enhance TORC2phosphorylation will find use as therapeutic agents in this setting.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E demonstrate that the CREB bZIP domain is required forcooperativity between cAMP and calcium signals. Specifically, FIG. 1Asummarizes the results of a transient transfection assay of HITinsulinoma cells transfected with the CREB-dependent EVX-1 luciferasereporter. Cells were treated with KCl (45 mM) and/or forskolin (10 μM)for six hours as indicated. The effect of treatment with the calcineurininhibitor, Cyclosporine A (CsA, 5 FM) is shown.

FIG. 1B presents quantitative PCR (Q-PCR) analysis of the CREB targetgene NR4A2 in MIN6 insulinoma cells exposed to glucose (20 mM) and/orexendin-4 (10 nM). The effect of the calcium channel antagonist,nifedipine, and CsA are shown.

FIG. 1C is a Western blot assay of phospho (Ser133) CREB levels in HITcells following treatment with KCl (45 mM), forskolin (10 μM), or bothtogether for 30 minutes. The effect of CsA treatment on levels ofphospho (Ser133) CREB is indicated.

FIG. 1D illustrates the effect of calcium and cAMP agonists on CREB:CBPcomplex formation. A mammalian two-hybrid assay was carried outemploying HIT cells transfected with GAL4-KID and KIX-VP16 expressionplasmids. The luciferase activity from cells co-transfected with GAL4luciferase reporter is shown. Cells were treated with forskolin and KCl(6 hours), alone and in combination, as indicated.

FIG. 1E illustrates the effect of KCl and forskolin on the activity offull-length GAL4-CREB and truncated GAL4-CREB ΔbZIP polypeptides lackingthe bZIP domain (amino acid residues 284-341) in transfected HIT cells.Treatment with CsA is shown.

FIGS. 2A-2D demonstrate that TORC2 is recruited to the promoter andmediates CREB target gene activation in response to CAMP and calciumsignals. Specifically, FIG. 2A summarizes the results of a transientassay of HIT cells co-transfected with CREB-dependent EVX-I luciferasereporter and TORC2 expression vector, or control empty vector, Cellswere treated with forskolin or KCl as indicated. The effect of CsA (5μM) is shown.

FIG. 2B illustrates the effect of TORC2 knockdown on induction of EVX-1reporter in response to cAMP. The left panel illustrates a transientassay of HEK293T cells co-transfected with non-specific or TORC2 RNAiplasmid plus EVX-1 reporter. The right panel illustrates the rescue ofactivity with TORC3 expression plasmid shown. The inset presents aWestern blot assay of endogenous TORC2 protein levels in TORC2 RNAi ornon-specific RNAi transfected cells.

FIG. 2C summarizes the results of transient transfection assays of HITcells expressing wild-type and mutant GAL4-CREB polypeptides that aredefective in either CBP (M1: Ser133Ala) or TORC (M2: Arg314Ala) binding.Comparable expression of wild-type and mutant GAL4-CREB polypeptides wasconfirmed by Western blot assay (not shown). The effect of forskolin (F)and KCl (K), either alone or in combination, on co-transfected GAL4luciferase reporter is shown. Treatment with CsA is indicated.

FIG. 2D presents the results of chromatin immunoprecipitation assay ofHEK293T cells using CREB, CBP, and TORC2 (T2) specific antisera. PCRamplification of the cAMP responsive NR4A2 promoter (5′) and 3′ flankingregion fragments from CREB, CBP, and TORC2 immunoprecipitates is shown.The effect of forskolin treatment (30 min) is indicated. Linearity ofthe PCR assay with decreasing DNA input levels is also indicated.

FIGS. 3A-3D demonstrate that cAMP promotes cytoplasmic to nucleartranslocation of TORC2. Specifically, FIG. 3A presents fluorescencemicroscopic analysis of Flag-tagged TORC2 in human ATYB1 fibroblaststreated with forskolin (10 μM) or control vehicle for 30 minutes asindicated.

FIG. 3B illustrates the effect of exportin inhibitor leptomycin B (LMB)on nuclear targeting of TORC2 in transfected ATYB1 fibroblasts, DAPIstaining is shown alongside to indicate nuclei.

FIG. 3C, top panel, illustrates the effect of Tyr282 to Phe mutagenesisin NES1 on TORC3 localization in control and forskolin treated ATYB1cells. Wild-type TORC3 staining in control cells is also shown. FIG. 3C,bottom panel, presents amino acid sequence alignments for NES1 and NES2motifs in TORC1, TORC2, and TORC3, relative to consensus NES motif shownbelow (SEQ ID NOS: 10-16, respectively, in order of appearance). ResidueTyr282 in TORC3 is highlighted.

FIG. 3D, top panel, presents a comparison of basal TORC2 (T2) and TORC3(T3) activities in HEK293T cells cotransfected with EVX-1 reporter.Comparable expression of TORC2 and TORC3 proteins was confirmed byWestern blot analysis (not shown). The effect of N-terminal srcmyristylation tag on TORC2 and TORC3 activities is shown. FIG. 3D,bottom panel, demonstrates the effect of adding the Tyr282Phe mutationon basal TORC3 activity, relative to empty vector control in HIT cellsco-transfected with EVX-1 reporter.

FIGS. 4A-4C demonstrate that cAMP and calcium promote TORC2dephosphorylation and nuclear entry. Specifically, FIG. 4A, left panel,presents a Western blot of nuclear (N) and cytoplasmic (C) fractionsfrom HEK293T cells transfected with Flag-tagged TORC2 (FLAG-T2) or emptyvector (ctrl). Compare the mobility of TORC2 immunoreactive bands.Endogenous CREB immunoreactivity is shown to verify the efficientfractionation of cytoplasmic and nuclear extracts. FIG. 4A, right panel,illustrates the effect of calf intestinal alkaline phosphatase (CIP)treatment on the electrophoretic mobility of cytoplasmic TORC2.

FIG. 4B illustrates the effect of co-stimulation with forskolin and KCl(F+K: 30 minutes) on endogenous TORC2 phosphorylation in HIT cells.Treatment with CsA is indicated.

FIG. 4C, top panel presents an SDS-PAGE analysis of ³²P-labeledFlag-TORC2 immunoprecipitates from transfected HIT cells incubated withinorganic ³²P. Control cells transfected with empty vector (vec) arealso indicated. Treatment with forskolin (F), KCl (K), or forskolin plusKCl are shown. The effect of CsA is indicated. FIG. 4C, middle panel,presents a Western blot assay of immunoprecipitated ³²P labeled TORC2protein from top panel. FIG. 4C, bottom panel, illustrates the relativelevels of TORC2 phosphorylation for each treatment, normalized to theimmunoprecipitated TORC2 protein levels shown.

FIGS. 5A-5D illustrate that 14-3-3 proteins associate with and repressTORC activity. Specifically, FIG. 5A, top panel, presents the results ofa coimmunoprecipitation assay of Flag-TORC1 with endogenous 14-3-3proteins in HEK293T cells. The effect of forskolin (F) treatment on theTORC1: 14-3-3 association is shown. The relative effect of Ser/Thrphosphatase PP1/PP2A (okadaic acid; OA) or CsA on TORC1: 14-3-3interaction is indicated. FIG. 5A, bottom left panel, is a Western blotassay of 14-3-3 proteins recovered from TORC or control 1 gGimmunoprecipitates, illustrating the effect of forskolin treatment onendogenous TORC: 14-3-3 complexes in PC12 and HEK293T cells. Inputlevels of 14-3-3 and TORC proteins are shown. Comparable recovery ofendogenous TORC proteins from TORC immunoprecipitates is indicated. FIG.5A, bottom right panel, illustrates the kinetics of TORC2 dissociationfrom endogenous 14-3-3 proteins in response to forskolin treatment ofHEK293T cells. The levels of Flag-tagged TORC 2 (FLAG-T2) recovered fromflag immunoprecipitates at each time point is shown below.

FIG. 5B summarizes the results of co-immunoprecipitation assays usingtruncated TORC2 polypeptides to define the 14-3-3 interaction site. Thedeletion endpoints in TORC2 are shown.

FIG. 5C illustrates the effect of 14-3-3 beta over-expression on EVX-1reporter activity in HEK293T cells co-transfected with wild-type TORC2,14-3-3 interaction defective (A56-547) TORC2, or empty vector.

FIG. 5D demonstrates that TORC2 binds to calcineurin. A pull-down assayof GST-calcineurin A (amino acid residues 1-347) with ³⁵S-labeledwildtype and mutant TORC2 polypeptides containing internal deletions areindicated.

FIGS. 6A-6D demonstrate that SIK2 (or related family members), snf1-likekinase, associates with and phosphorylates TORC2. Specifically, FIG. 6Ademonstrates that TORCs associate with a cytoplasmic protein kinaseactivity. In vitro kinase assays of endogenous TORC (1P:TORC) andtransfected Flag-tagged TORC2 (1P:FLAG) immunoprecipitates were preparedfrom nuclear or cytoplasmic fractions of control and forskolin treatedcells as shown. ³²P-labeled bands (left) and corresponding proteinlevels by Western blot assay (right) are indicated.

FIG. 6B summarizes immunoprecipitation assays of HEK293T cellsco-transfected with expression vectors for SIK2 and Flag-tagged TORC2.Western blot assays of SIK2 recovered from Flag immunoprecipitates usinganti-SIK2 antiserum are shown.

FIG. 6C identifies phosphopeptides recovered from Flag-tagged TORC2immunoprecipitates identified by MSMS analysis (SEQ ID NOS: 17-27,respectively, in order of appearance). Amino acid endpoints andphosphorylated residues are indicated. Consensus SIK2 phosphorylationsite shown: φ=hydrophobic; B=basic, X=any amino acid.

FIG. 6D demonstrates that SIK2 phosphorylates TORC2 at Ser171. An invitro kinase assay of wild-type and Ser171Ala GST TORC2 polypeptides(amino acid residues 162-179) was conducted using purified SIK2 asindicated. The effect of SIK2 (or related family members) onphosphorylation of GST alone or GST-snide 2 containing a consensus SIK2phosphorylation site (PLARTLSVAGLPGKK; SEQ ID NO:1) is shown. Inputlevels of individual GST proteins (CBB) are shown below.

FIGS. 7A-7C demonstrate that TORC2 is retained in the cytoplasm underbasal conditions via SIK2 (or related family members)-dependentphosphorylation at Ser171. Specifically, FIG. 7A presents a Western blotanalysis of total and phospho (Ser587)-SIK2 levels in COS-7 cellstransfected with wild-type and PKA phosphorylation defective Ser587Alamutant SIK2 expression vector. The effect of forskolin treatment isshown.

FIG. 7B, left panel, presents a comparison of wild-type, PKAphosphorylation defective (Ser587Ala), and kinase-inactive (Lys49Met)SIK2 constructs on EVX-1 reporter activity in control and forskolintreated HEK293T cells. FIG. 7B, right panel, illustrates the effect ofwild-type and Scr171Ala mutant TORC2 polypeptides on EVX-1 reporteractivity in control and forskolin stimulated HEK293T cells.

FIG. 7C, top panels, illustrate the effect of SIK2 (or related familymembers) on TORC2 subcellular localization. Immunofluorescencemicroscopy is presented for ATYB1 cells transfected with FlagtaggedTORC2 plus wild-type or PKA phosphorylation defective SIK2 (Ser587Ala)as indicated. DAPI staining is shown below each panel. FIG. 7C, bottompanels, illustrate the effect of Ser171Ala mutagenesis on cellularlocalization of TORC2. Treatment with forskolin is indicated.

FIG. 8 presents a model for cooperative induction of cellular genes inresponse to cAMP and calcium signals in insulin producing beta cells.SIK2 (and related family members) promotes Ser171 phosphorylation andassociation of TORC2 with 14-3-3 proteins. cAMP and calcium agonistsactivate TORC2 via cooperative effects on TORC2 dephosphorylation andnuclear entry. cAMP inhibits TORC2 associated activity of SIK2 andrelated family members, whereas calcium signals promote calcineurindependent dephosphorylation of TORC2. Nuclear TORC2 stimulates CREBactivity via an interaction with the bZIP domain of CREB. In parallel,cAMP stimulates phosphorylation of CREB at Ser133 and recruitment of thehistone acetylase CBP to the promoter.

FIG. 9 illustrates the time course (in minutes) of CREB Ser133phosphorylation in HIT cells following treatment with forskolin (10 μM)or KCl (45 mM). The effect of cyclosporine A (CsA: 5 μM) is shown.Immunoreactive bands correspond to phospho (Ser133) CREB (top) andphospho (Ser63) ATF1 (bottom).

FIG. 10 presents the characterization of a TORC interaction defectivemutant CREB polypeptide. A pull-down assay of ³⁵S-labeled TORC1 wascarried out with wild-type and mutant GST CREB hZIP (amino acid residues284-341) polypeptides. The effect of alanine substitutions at polarresidues in the leucine zipper domain on TORC binding are shown. Bindingof TORC to itself via N-terminal coiled-coil domain (TORC1-129) is shownfor comparison. Coomassie stained gel showing comparable input levels ofGST-CREB bZIP proteins was used for the pull-down assays.

FIG. 11 presents the results of transient assays of HEK293T cellstransfected with flag-tagged wild-type, Arg314Ala (R314A), and Glu319Ala(E319A) CREB plus TORC1 expression vector. Luciferase activity obtainedfrom co-transfected EVX-1 reporter plasmid is shown. Comparable bindingof wild-type and mutant Arg314Ala (R314A) CREB to the CRE was verifiedby gel mobility shift assay (not shown).

FIG. 12 indicates the relative importance of CREB:CBP and CREB:TORCcomplexes for transcriptional activation in response to CAMP and calciumsignals in PC12 cells. Transient assay was conducted with PC12 cellsco-transfected with wild-type, CBP interaction defective (M1:Ser133Ala), TORC interaction defective (M2: Arg314Ala), or CBP and TORCdefective (M1/M2) GAL4-CREB expression vectors as indicated. Theactivity was determined for cotransfected GAL4 luciferase reporter incells treated with forskolin (10 μM), KC 1 (45 mM), or forskolin plusKCl for four hours as shown.

FIG. 13, top panels, illustrates the cellular localization of endogenousTORC proteins in ATYB1 cells using TORC specific antiserum. The effectof forskolin (10 μM, 30 minutes) and leptomycin B (LMB, 10 ng/ml, 2 hr)treatment is shown. DAPI staining is shown below to visualize thenuclei. FIG. 13, bottom panel, illustrates the effect of LMB treatmenton TORC1 localization in ATYB1 cells transfected with flag-tagged TORC1expression vector. TORC1 localization was followed using anti-flagantiserum. DAP1 staining is shown alongside.

FIG. 14 identifies nuclear localization (NLS) and nuclear export (NES)signals in TORC. Summary in the right margin thereof shows thepredominant cellular location (Nuclear:N, Cytoplasmic:C) of TORC2polypeptides fused to green fluorescent protein (GFP). TORC2 amino acidendpoints for each fusion protein shown.

FIG. 15 provides the characterization of optimal phosphorylation sitesfor SIK2. Potential SIK2 substrates identified in database search weretested by in vitro kinase assay with purified SIK2. The table shows thesubstrates tested and the relative stoichiometry of phosphorylation. Theoptimal motif for SIK2 mediated phosphorylation is shown. FIG. 15discloses SEQ ID NOS: 1 and 28-53, respectively, in order of appearance.

FIGS. 16A-16E illustrate the effect of fasting and feeding signals onactivation of CREB:TORC and CREB:CBP pathways in liver. Specifically,FIG. 16A presents an immunohistochemical analysis of CREB and phospho(Ser133) CREB staining on liver sections from mice 10 minutes followingintraperitoneal (IP) injection with insulin, glucagon, or vehicle (PBS).DAPI staining is shown to highlight nuclei.

FIG. 16B presents a Western blot analysis of phospho (Ser133) CREB andCREB levels in liver extracts prepared from the same three treatmentgroups as described above.

FIG. 16C presents an immunohistochemical analysis of TORC2 localizationin liver sections from mice 10 minutes following the sameintraperitoneal (IP) injections as described above with respect to FIG.16A. DAPI staining is shown to highlight nuclei.

FIG. 16D presents a Western blot assay of HA-TORC2 immunoprecipitatesprepared from whole liver extracts of treatment groups described abovewith anti-HA antiserum. Western blotting with phospho (Ser171) specificand non-discriminating TORC2 antiserum is shown. The top band in theglucagon treated sample corresponds to Ser171-phosphorylated (PTORC2).

FIG. 16E presents the results of a Chromatin Immunoprecipitation (ChIP)Assay of liver extracts from mice 10 minutes following IP glucagon orinsulin administration. The recruitment of TORC2 to CREB target genes(PEPCK, G6Pase) in liver is demonstrated. FDPS, CREB target gene isinduced only in the fed state (FDPS) in liver. Levels of TORC2recruitment to each gene were determined by Q-PCR analysis.

FIGS. 17A-17F demonstrate that TORC2 is required for hepaticgluconeogenesis during fasting. Specifically, FIG. 17A illustrates theeffect of TORC2 on induction of gluconeogenic genes (PEPCK, PGC-1α,G6Pase) by FSK (10 μM, 2 hr) in cultured primary rat hepatocytesinfected with TORC2 or control GFP adenovinus as indicated.

FIG. 17B demonstrates that TORC2 stimulates gluconeogenic genes viaCREB. The effect of dominant negative A-CREB (AC) adenovirus on G6PasemRNA levels is illustrated in cells co-infected with GFP or TORC2adenovirus and treated with FSK as indicated.

FIG. 17C summarizes the effect of TORC on glucose output from primaryrat hepatocytes in response to fasting and feeding signals. Cells wereinfected with TORC1, TORC2, or control GFP adenovirus and then treatedwith FSK plus dexamethasone (FSK/DEX) for four hours. The effect ofinsulin on glucose output is shown.

FIG. 17D summarizes the effect of TORC2 over-expression on fastingglucose metabolism. Blood glucose levels in control (GFP) and TORC2adenovirus injected mice is shown, taken sequentially after 7 and 24hours of fasting (n=3). Below the graph presented in FIG. 17D, a Westernblot analysis of liver extracts from control and TORC2 adenovirusinfected mice is presented, showing the relative levels of endogenousand adenovinus expressed HA-TORC2 (denoted by an arrow).

FIG. 17E summarizes the effect of acute TORC2 knockdown on fastingglucose levels in mice (n=3). Mice were infected with TORC2 RNAi orunspecific (US) RNAi adenovirus. The inset presents a Western blot assayof TORC2 levels in primary hepatocytes infected with unspecific on TORC2RNAi adenovirus.

FIG. 17F presents a Q-PCR analysis of hepatic mRNAs from TORC2 deficient(TORC2 RNAi) and control (US) mice. The levels of gluconeogenic (PEPCK,PGC-1α, G6Pase, PC) and mitochondrial (Cox4, Cyt-C) gene expression areshown.

FIGS. 18A-18H illustrate that the induction of the AMP kinase familymember SIK1 by CREB during fasting attenuates the gluconeogenic program.Specifically, FIG. 18A illustrates the time course of PEPCK geneexpression in primary rat hepatocytes following exposure to FSK. mRNAlevels were determined by Q-PCR analysis.

FIG. 18B illustrates the effect of CHX on levels of phospho (Ser171)TORC2 in primary rat hepatocytes treated with glucagon over a variety oftime points, shown in hours. Western blot assay indicates the levels ofTORC2, phospho (Ser171) TORC2 and CREB.

FIG. 18C illustrates the effect of CHX pretreatment on induction ofgluconeogenic genes (PEPCK, PGC-1α, G6Pase) by glucagon in primary rathepatocytes. Relative mRNA levels are shown.

FIG. 18D summarizes the effect of fasting on mRNA levels for SIK1, SIK2,and SIK3 genes in liver by Q-PCR analysis (n=3).

FIG. 18E presents a Western blot analysis of SIK1 protein levels inwhole liver extracts from mice under ad libitum, fasting, or refedconditions. CREB levels are shown for comparison.

FIG. 18F summarizes the effect of FSK treatment on SIK1 mRNA levels inprimary rat hepatocytes. Cells were infected with control (GFP) ordominant negative A-CREB adenovirus as indicated.

FIG. 18G summarizes the effect of co-transfected PKA, dominant negativeA-CREB, or control (empty) expression vector on SIK1 luciferase reporteractivity in transiently transfected HepG2 hepatocytes.

FIG. 18H, top, provises a schematic of a SIK1 promoter, showing thepresence of two CRE sites at positions indicated relative to thetranscriptional start site. FIG. 18H, bottom, presents the results of aChromatin Immunoprecipitation (ChIP) assay of CREB immunoprecipitatesprepared from SV40-transformed mouse hepatocytes, showing recovery ofSIK1 promoter or negative control ACT B promoter, which lacks consensusCREB binding site. Genomic DNA input (In) levels (1%) are shown.

FIGS. 19A-19F demonstrate that SIKs inhibit hepatic gluconeogenesis viaphosphorylation of TORC2 at Ser171. Specifically, FIG. 19A, top,presents a Western blot showing the effect of US and SIK1 RNAiadenoviruses on the levels of SIK1 protein in rat hepatocytes. FIG. 19A,bottom, presents a Western blot assay with nondiscriminating TORC2antiserum, showing the effect of SIK1 RNAi adenovirus on TORC2dephosphorylation (compare P-TORC2 and TORC2 bands) following exposureof primary rat hepatocytes to glucagon (Gluc). The arrow identifiesphosphorylated TORC2.

FIG. 19B summarizes the effect of SIK1 RNAi on gluconeogenic geneexpression in primary rat hepatocytes, presenting Q-PCR analysis showingthe relative induction of PEPCK, PGC-1α, and G6Pase mRNAs by glucagon incells infected with control (US) or SIK1 RNAi expressing adenoviruses.

FIG. 19C summarizes the effect of SIK1 and SIK2 relative to control(GFP) adenovirus injection on fasting glucose levels in mice (n=4).

FIG. 19D presents a Q-PCR analysis of gluconeogenic gene expression(PEPCK, G6Pase, PGC-1α) in livers of fasted mice infected with control(GFP), SIK1, or SIK2 Adenovirus.

FIGS. 19E and 19F demonstrate the role of Ser171 in TORC2 for SIK1inhibition of gluconeogenic genes. Specifically, the effect of SIK1 onthe induction of gluconeogenic PGC-1α (see FIG. 19E) and PEPCK (see FIG.19F) genes by wild-type and Ser171Ala TORC2 is illustrated in primaryrat hepatocytes.

FIGS. 20A-20E demonstrate that the energy sensing AMPK (AMP-activatedprotein kinase) pathway regulates TORC2 activity in liver. Specifically,FIG. 20A illustrates the relative phosphorylation of wild-type orSer171Ala mutant recombinant GST-TOR2 (161-181) compared to GST only oroptimal AMPK peptide substrate (SAMS) by activated AMPK in vitro.Addition of AMP to reactions is shown. The relative incorporation ofγ³²P-ATP by each substrate is indicated.

FIG. 20B demonstrates the effect of the AMP analog A1CAR (1 mM) onphosphorylation of endogenous TORC2 in primary rat hepatocytes. Cellswere exposed to A1CAR and FSK for 30 minutes. Lower mobility bands(arrow) indicate phosphorylated TORC2 polypeptides.

FIG. 20C illustrate the effect of A1CAR and SIK1 on cellularlocalization of wild-type or Ser171 Ala TORC2 in primary rathepatocytes. Cells were infected with adenoviruses for HA-taggedwild-type or mutant TORC2 and SIK1 as indicated. Infected cells wereexposed to FSK and AICAR for 30 minutes. TORC2 localization was examinedwith anti-HA antiserum. Cells were counterstained with DAPI to visualizenuclei.

FIG. 20D summarizes the effect of A1CAR on expression of gluconeogenicgenes (PEPCK, PGC-1α) in primary rat hepatocytes. Cells infected withmutant Ser171 Ala TORC2 (171) adenovinus indicated. Treatment with AICARand FSK is shown.

FIG. 20E illustrates an auto-regulatory loop which controlsgluconeogenic gene expression in liver. Activation of the cAMP pathwayby glucagon triggers expression of the gluconeogenic program via acuteSer171 dephosphorylation and activation of TORC2. At late times afterglucagon stimulation TORC2 activity is attenuated via CREB-mediatedinduction of SIK1, which in turn rephosphorylates TORC2 at Ser171. Inresponse to ATP depletion, TORC2 activity is also inhibited by AMPK(AMP-activated protein kinase) mediated phosphorylation at Ser171.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided methods ofscreening test compounds to determine if such compounds are capable ofenhancing islet cell activity and/or survival. Invention methodscomprise determining the effect of test compound on one or more of:

-   -   the transport of a Transducer Of Regulated CREB (TORC) from the        cytoplasm into the nucleus of an islet cell,    -   the kinase activity of a snf1-like kinase,    -   the interaction between a Transducer Of Regulated CREB (TORC)        and a member of the 14-3-3 family of proteins, or    -   the level of phosphorylation of a Transducer Of Regulated CREB        (TORC),    -   wherein one or more of the following, in the presence of test        compound, is indicative of a compound which is capable of        enhancing islet cell activity and/or survival:    -   enhanced transport of TORC from the cytoplasm into the nucleus        of an islet cell,    -   reduced kinase activity of the snf1-like kinase,    -   disruption of the interaction between TORC and a member of the        14-3-3 family of proteins, or    -   a reduction in the level of phosphorylation of TORC.

In accordance with one embodiment of the present invention, there areprovided methods of screening test compounds to determine if suchcompounds are capable of enhancing islet cell activity and/or survival.Invention methods comprise determining the effect of test compound onthe transport of a Transducer Of Regulated CREB (TORC) from thecytoplasm into the nucleus of an islet cell, wherein enhanced transportof TORC from the cytoplasm into the nucleus of an islet cell in thepresence of test compound is indicative of a compound which is capableof enhancing islet cell activity and/or survival.

As employed herein, “islet cell activity” refers to the role islet cellsplay in the regulation of blood glucose levels. For example, beta isletcells produce and secrete insulin, which acts to decrease blood glucoselevels by mediated cell absorption of glucose. Failure (e.g., celldeath) of beta islet cells (and hence, the ability to produce insulin)has been implicated, for example, in the development of type II diabetesin obese individuals. Similarly, in type I diabetes, beta islet cellsare depleted by autoimmune attack thereon. In contrast to the role ofbeta islet cells, alpha islet cells secrete glucagon, which increasesblood glucose levels by stimulation of cellular production and releaseof glucose.

As employed herein, “survival” of islet cells refers to the continuedviability of islet cells whether in native host tissue, or upontransplantation, the ability of islet cells to resist the factors whichlead to cell death (and hence type II diabetes), the ability of isletcells to resist the factors which lead to autoimmune attack thereon, andthe like.

As readily recognized by those of skill in the art, a number ofTransducers Of Regulated CREB (TORCs) are known. Indeed, severalisoforms have been identified (e.g., TORC1, TORC2, TORC3, and the like).TORCs have been shown to accumulate in the nucleus in response toincreased intracellular calcium or cAMP, contributing to activation ofCRE-dependent transcription.

In accordance with another embodiment of the present invention, thereare provided methods of screening test compounds to determine if suchcompounds are capable of enhancing islet cell activity and/or survival.Invention methods comprise determining the effect of test compound onthe kinase activity of a sucrose-nonfermenting-1 (snf1)-like proteinkinase, wherein reduced kinase activity of the snf1-like kinase in thepresence of said test compound is indicative of a compound which iscapable of enhancing islet cell activity and/or survival.

As readily recognized by those of skill in the art, a number ofsnf1-like kinases are known, e.g., SIK1, SIK2, SIK3, and the like. TheseSIK proteins are serine/threonine kinases and are members of anAMP-activated protein kinase family. Salt-inducible kinase-1 (SIK1) wasfirst isolated from the adrenal glands of rats on a high salt diet. SIK1is primarily expressed in rat adrenal gland and may play a role inregulating steroidogenic gene expression. SIK2 and SIK3 weresubsequently cloned and exhibit adipose-specific and ubiquitousexpression, respectively. SIK2 phosphorylates cytoplasmic TORC atSer171, thereby mediating the phosphate-dependent interaction of TORCand 14-3-3. This results in a repression of TORC translocation to thenucleus and a decrease in TORC-mediated CREB activity.

In accordance with still another embodiment of the present invention,there are provided methods of screening test compounds to determine ifsuch compounds are capable of enhancing islet cell activity and/orsurvival. Invention methods comprise determining the effect of testcompound on the interaction between a Transducer Of Regulated CREB(TORC) and a member of the 14-3-3 family of proteins, wherein disruptionof the interaction between TORC and a member of the 14-3-3 family ofproteins in the presence of test compound is indicative of a compoundwhich is capable of enhancing islet cell activity and/or survival.

As readily recognized by those of skill in the art, 14-3-3 proteinscomprise a family of phosphoserine/phosphothreonine binding proteins. Atleast seven isoforms of 14-3-3 (β, γ, ε, σ, ζ, τ and η) have beendescribed in mammalian cells. 14-3-3 proteins form homodimers andheterodimers capable of interacting with other cellular proteins. Thephosphoserine/phosphothreonine-binding activity of 14-3-3 enables thesemolecules to interact with a wide variety of other cellular proteins andthereby contribute to the regulation of a number of important cellularprocesses (e.g., metabolism, apoptosis, cell cycle control, and thelike). 14-3-3 proteins repress TORC translocation to the nucleus via aphosphate-dependent interaction with TORC. Disruption of suchinteractions (e.g, via dephosphorylation of TORC) allows TORC totranslocate to the nucleus and increase TORC-mediated CREB activity.

In accordance with a further embodiment of the present invention, thereare provided methods of screening test compounds to determine if suchcompounds are capable of enhancing islet cell activity and/or survival.Invention methods comprise determining the effect of test compound onthe level of phosphorylation of a Transducer Of Regulated CREB (TORC),wherein a reduction in the level of phosphorylation of TORC in thepresence of test compound is indicative of a compound which is capableof enhancing islet cell activity and/or survival.

There are a variety of positions on the TORCs which can bephosphorylated, e.g., at a position comparable to Ser171 of TORC2, andthe like.

In accordance with a further embodiment of the present invention, thereare provided methods of screening test compounds to determine if suchcompounds are capable of promoting CREB-mediated gene expression inislet cells. Invention methods comprise determining the effect of testcompound on one or more of:

-   -   the transport of a Transducer Of Regulated CREB (TORC) from the        cytoplasm into the nucleus of an islet cell,    -   the kinase activity of a snf1-like kinase,    -   the interaction between a Transducer Of Regulated CREB (TORC)        and a member of the 14-3-3 family of proteins, or    -   the level of phosphorylation of a Transducer Of Regulated CREB        (TORC),    -   wherein one or more of the following, in the presence of test        compound, is indicative of a compound which is capable of        promoting CREB-mediated gene expression in islet cells:    -   enhanced transport of TORC from the cytoplasm into the nucleus        of an islet cell,    -   reduced kinase activity of the snf1-like kinase,    -   disruption of the interaction between TORC and a member of the        14-3-3 family of proteins, or    -   a reduction in the level of phorphorylation of TORC.

In accordance with still another embodiment of the present invention,there are provided methods of screening test compounds to determine ifsuch compounds are capable of promoting CREB-mediated gene expression inislet cells. Invention methods comprise determining the effect of testcompound on the transport of a Transducer Of Regulated CREB (TORC) fromthe cytoplasm into the nucleus of an islet cell, wherein enhancedtransport of TORC from the cytoplasm into the nucleus of an islet cellin the presence of test compound is indicative of a compound which iscapable of promoting CREB-mediated gene expression in islet cells.

In accordance with yet another embodiment of the present invention,there are provided methods of screening test compounds to determine ifsuch compounds are capable of promoting CREB-mediated gene expression inislet cells. Invention methods comprise determining the effect of testcompound on the kinase activity of a snf1-like kinase, wherein reducedkinase activity of the snf1-like kinase in the presence of said testcompound is indicative of a compound which is capable of promotingCREB-mediated gene expression in islet cells.

In accordance with another embodiment of the present invention, thereare provided methods of screening test compounds to determine if suchcompounds are capable of promoting CREB-mediated gene expression inislet cells. Invention methods comprise determining the effect of testcompound on the interaction between a Transducer Of Regulated CREB(TORC) and a member of the 14-3-3 family of proteins, wherein disruptionof the interaction between TORC and a member of the 14-3-3 family ofproteins in the presence of test compound is indicative of a compoundwhich is capable of promoting CREB-mediated gene expression in isletcells.

In accordance with still another embodiment of the present invention,there are provided methods of screening test compounds to determine ifsuch compounds are capable of promoting CREB-mediated gene expression inislet cells. Invention methods comprise determining the effect of testcompound on the level of phosphorylation of a Transducer Of RegulatedCREB (TORC), wherein a reduction in the level of phorphorylation of TORCin the presence of test compound is indicative of a compound which iscapable of promoting CREB-mediated gene expression in islet cells.

In accordance with yet another embodiment of the present invention,there are provided methods of screening test compounds to determinewhether such compounds effect transport of a Transducer Of RegulatedCREB (TORC) from the cytoplasm into the nucleus of an islet cell.Invention methods comprise determining the effect of test compound onthe transport of TORC from the cytoplasm into the nucleus of an isletcell.

As readily recognized by those of skill in the art, a test compoundwhich effects transport of TORC from the cytoplasm into the nucleus of acell can either enhance such transport, or reduce such transport.

There are a variety of cells in which TORC transport is important, e.g.,islet cells (e.g., alpha and beta islet cells), muscle cells (e.g.,cardiac muscle), liver cells, adipose cells, neuronal cells, and thelike.

In accordance with a further embodiment of the present invention, thereare provided methods of screening test compounds to determine whethersuch compounds effect the kinase activity of an snf1-like kinase.Invention methods comprise determining the effect of test compound onthe kinase activity of an snf1-like kinase.

As readily recognized by those of skill in the art, a test compoundwhich effects the kinase activity of an snf1-like kinase can eitherenhance such activity, or reduce such activity.

In accordance with another embodiment of the present invention, thereare provided methods of screening test compounds to determine whethersuch compounds effect the interaction between a Transducer Of RegulatedCREB (TORC) and a member of the 14-3-3 family of proteins. Inventionmethods comprise determining the effect of test compound on theinteraction between TORC and a member of the 14-3-3 family of proteins.

A test compound which effects the interaction between TORC and a memberof the 14-3-3 family of proteins can either enhance or disrupt suchinteraction.

In accordance with still another embodiment of the present invention,there are provided methods of screening test compounds to determinewhether such compounds effect the level of phosphorylation of aTransducer Of Regulated CREB (TORC). Invention methods comprisedetermining the effect of test compound on the level of phosphorylationof TORC. Optionally, the above-described method can be carried out inthe further presence of a phosphatase such as calcineurin.

As readily recognized by those of skill in the art, a test compoundwhich effects the level of phosphorylation of TORC can either enhance orreduce the level of phosphorylation of TORC.

In accordance with a further embodiment of the present invention, thereare provided compounds identified by any of the above-described methods.

In accordance with yet another embodiment of the present invention,there are provided methods for enhancing islet cell activity and/orsurvival, said method comprising enhancing the transport of a TransducerOf Regulated CREB (TORC) from the cytoplasm into the nucleus of an isletcell.

As readily recognized by those of skill in the art, transport of TORCfrom the cytoplasm into the nucleus of an islet cell can be enhanced ina variety of ways, e.g., by blocking activation of SIK2 (or relatedfamily members), by disrupting the interaction between TORC and a memberof the 14-3-3 family of proteins, by dephosphorylating TORC, and thelike. As readily recognized by those of skill in the art, this can beaccomplished in a variety of ways, e.g., by administering an effectiveamount of a compound identified by any of the above-described methods toa subject in need thereof.

Described herein is a new pathway that operates in parallel with thehistone acetylase coactivators CBP/P300 to activate cellular genes inresponse to cAMP and calcium signals (see FIG. 8). Despite theircooperative effects on cellular gene expression, calcium and cAMP do notpromote either CREB Ser133 phosphorylation or CREB:CBP complex formationin a synergistic fashion. Indeed, knocking mice with point mutations inthe KIX domain of CBP and P300 that disrupt the CREB interaction displayonly modest changes in cAMP responsiveness (Kasper et al., 2002),consistent with the existence of a distinct pathway that also mediatescellular gene activation via CREB.

The CREB:TORC pathway is activated in response to extracellular signals;it does not require Ser133 phosphorylation but rather operates throughthe CREB bZIP domain, a region which has been found to contributefunctionally to cAMP and calcium signaling in excitable cells (Boni etal., 1995b; Sheng et al., 1991), The importance of this domain fortarget gene activation may explain in part why CREB homodimerizesselectively with related family members (CREB1, ATF1, CREM) and not withother bZIP proteins (Newman and Keating, 2003).

TORCs fulfill a number of criteria for coactivators that mediatecooperativity between cAMP and calcium signals. First, they stimulateCREB activity through a bZIP domain interaction; point mutants thatdisrupt this interaction compromise cellular responses to both cAMP andcalcium agonists. Indeed, TORC2 is recruited to the promoter in asignal-dependent manner, and knockdown of TORC2 disrupts induction ofCREB target genes in response to cAMP.

Disrupting the CREB:CBP interaction by mutagenesis of the Ser133phosphoacceptor site in CREB compromises cAMP inducibility, but has noeffect on either the costimulatory actions of cAMP and calcium secondmessengers nor on the ability of CsA to block this cooperativity. Theimportance of the bZIP domain but not phospho (Ser133) for calciumdependent transcription may explain in part why CREB remains active inthis setting even though calcium signals trigger its phosphorylation atsites (Ser142 and 143) that are inhibitory for the CREB: CBP interaction(Kornhauser et al., 2002).

Without wishing to be bound by any theory, the putative mechanismunderlying TORC activation is reminiscent of the cytoplasmic family ofNFAT transcription factors (Crabtree and Olson, 2002; Hogan et al.,2003). Indeed, TORCs and NFATs are both maintained in the cytoplasmunder basal conditions via phosphorylation at sites that promote aninteraction with 14-3-3 proteins. Both sets of proteins aredephosphorylated in response to calcium signals via a direct associationwith the calcium dependent phosphatase calcineurin. Indeed, thecalcineurin binding site in TORC contains a sequence (TORC2: amino acid248-PGINIF; SEQ ID NO:2) that resembles the consensus calcineurininteraction motif identified for NFATs (PXIXIT; SEQ ID NO:3) (Aramburuet al., 1998), although the importance of this site for theTORC:calcineunin interaction was not addressed. Like NFATs, bindingsites for calcineunin and 14-3-3 proteins on TORC appear to clusterwithin a regulatory domain that also contains nuclear import and exportsignals. The Ser171 SIK2 phosphorylation site in TORC2 is part of apotential mode 2 binding site for 14-3-3 (RXXXpSXP; SEQ ID NO:4). Basedon its proximity to TORC2 NLS motifs (aa. 1-147), Ser171 is believed toperform a gatekeeper function, masking NLS motifs from the importmachinery in a manner comparable to NFAT (Okamura et al., 2000) andother proteins. It is of note that TORC2 is also phosphorylated atnumerous additional sites in addition to Ser171, and these may alsocontribute to TORC2 regulation.

cAMP and calcium promote TORC2 dephosphorylation cooperatively in betacells via their effects on TORC2 associated Ser/Thr kinase andphosphatase activities. Treatment with the calcineurin inhibitor CsAreverses the effects of cAMP and calcium on TORC dephosphorylation andCREB target gene activation. Conversely, cAMP promotes TORC activationby disrupting the TORC associated ser/thr protein kinase SIK2 (andrelated family members).

SIK2 is a member of a larger family of at least 13 AMPK (AMP-activatedprotein kinase) related kinases that includes 3 SIKs (SIK1, SIK2, SIK3),4 MARKs (MARK-1, MARK-2, MARK-3/PAR1A, MARK-4) as well as AMPK-α1, andAMPK-α2. The AMPK related family of kinases is activated throughphosphorylation by LKB-1, a tumor suppressor that is mutated in PeutzJehgers syndrome (PJS), a familial disorder characterized by multiplecolon polyps and an increased incidence of colon and other cancers(Carling, 2004; Lizcano et al., 2004; Shaw et al., 2004; Woods et al.,2003). LKB-1 phosphorylates AMPK family members, including SIK2, at aconserved Thr in the T-loop that stimulates their kinase activity morethan 50-fold (Carling, 2004). Loss of LKB-1 would be predicted toactivate CREB target gene expression by reducing levels of SIK2 (orrelated family member)-dependent TORC2 phosphorylation. Remarkably,TORC1 has been described as part of a fusion protein with the Notchco-activator mastermind like 2 (MAML2) that arises from a chromosomaltranslocation in mucoepidermoid carcinomas of the salivary and bronchialglands (Enlund et al., 2004; Tonon et al., 2003). Notably theTORC1-MAML2 fusion gene contains the CREB binding domain of TORC1 (aa.1-42) but lacks the central TORC regulatory domain that isphosphorylated by SIK2 (or related family members). Correspondingly, theTORC-MAML2 fusion gene is constitutively nuclear and displays high basalactivity on CREB target genes (Conkright et al., 2003a; Tonon et al.,2003). The results presented herein are consistent with the proposedmechanism whereby the loss of LKB-1 activity in PJS similarly promotesoncogenic transformation in part by activating the CREB:TORC pathway.

Elevations in circulating glucose and GLP-1 promote islet survival inpart through their cooperative effects on CREB activity. Calcineurininhibitors such as FK506 and CsA have been found to cause β cell failureand diabetes with high frequency in organ transplant recipientsreceiving chronic immunosuppressive therapy (Al-Uzri et al., 2001;Filler et al., 2000). Based on their ability to interfere with glucoseand incretin signaling to TORC, these calcineurin inhibitors may promoteislet cell death in part by blocking CREB target gene activation. Inthis regard, the development of specific SIK antagonists is expected toimprove islet cell function and offer useful therapy for insulinresistant individuals.

The effects of SIK2 and related family members on cellular generegulation by CREB is likely to extend to other electrically excitabletissues. Both SIKs and TORCs are highly expressed in the brain, forexample, an area where CREB appears to function in higher orderprocesses such as learning and memory. Targeted disruption of SIK andTORC genes in these and other tissues will enable one to mediateresponses to extracellular signals.

The invention will now be described in greater detail with reference tothe following non-limiting examples. See also Screaton et al., 2004 andKoo et al., 2005, the entire contents of each of which is herebyincorporated by reference.

EXAMPLES Chemicals

LMB and Exendin-4 (Sigma, Saint Louis, Mo.) were used at 10 ng/ml and 10nM, respectively. Nifedipine (10 uM), Cyclosporine A (CsA, 5 μM) andokadaic acid, (OA, 100 nM) were from Calbiochem (San Diego, Calif.). ³²Porthophosphate in 0.02 N HCl was from ICN.

Cell Culture:

HEK293T cells were cultured in DME+10% FBS+pen/strep. HIT-T15 cells werecultured in F12/K medium with 10% HS+2.5% FBS+pen/strep. MIN6 werecultured in DME with 10% FBS+pen/strep and 50 μM beta-mercaptoethanol.

Quantitative PCR:

Cells were treated with cAMP agonist (Forskolin, 10 μM) and KCl (45 mM)or vehicle control. For glucose experiments, MIN6 cells were starvedovernight in DME containing 2.75 mM glucose plus 16 mM mannitol and 10%FBS. The next day the medium was changed for the indicatedtimes±inhibitor. Total RNA from treated MIN6 or HIT cells was extractedusing an RNeasy mini-kit (Qiagen, Valencia, Calif.). 500 ng-1 μg oftotal RNA was used for generating cDNA with Superscript II enzyme(Invitrogen, Carlsbad, Calif.). cDNAs were analyzed by quantitative PCRusing SYBR green PCR kit and an ABIPRISM 7700 Sequence detector (PerkinElmer, Foster City, Calif.). All PCR data for CREB target NR4A2(Conkright et al., 2003b) was normalized to ribosomal L32 or 36B4expression in the corresponding sample.

Antisera, Western Blot Analysis, Immunoprecipitation and GST-Pulldown:

Rabbit pan-TORC (raised against 1-42 of human TORC1) and TORC2 selectiverabbit polyclonal antiserum (raised against amino acids 454-607 of mouseTORC2) were generated as previously described (Conkright et al.,2003a)). Whole cell protein was extracted from cultured HEK293T, HIT,and MIN6 cells in Laemmli SDS sample buffer. Alternatively, whole cellprotein was extracted from liver tissue or primary hepatocytes inSDS-urea-lysis buffer. 10-20 μg of protein was separated by 8, 10 or4-20% SDS-PAGE and transferred to PVDF membrane (Millipore Corp.,Bedford, Mass.). Alternatively, 50-100 μg of protein was loaded onto a6% SDS-polyacrylamide gel and blotted onto nitrocellulose membrane(Schleicher & Schuell, Keene, N.H.). Chromatin immunoprecipitation andco-immunoprecipitation experiments were performed as described(Conkright et al., 2003a).

Western blot analyses were conducted on whole cell, cytoplasmic andnuclear extracts with the following rabbit polyclonal antisera: phospho(Ser133) specific affinity purified 5322 and phospho (Ser133) specificantibody from Rockland Immunochemicals, Gilbertsville, Pa.),non-discriminating CREB (244), phosphor (Ser171) CREB, TORC2, pan-TORC,SIK1 and SIK2. 14-3-3 and Hsp90 antibodies were from Santa Cruz (SantaCruz, Calif.). FLAG-M2 (Sigma) and GAL4 (Santa Cruz) monoclonalantibodies were used at 1:2000 and 1:1000 dilutions, respectively. Forquantitative western blotting, goat anti-mouse-800 nm fluorophoreconjugate to detect FLAG-M2 was used at 1:40,000 dilution according tomanufacturer's instructions (Licor) prior to analysis using the Odysseydetection system. GST-pulldowns were performed as described (Asahara etal., 2001).

For immunoprecipitation, whole cell protein was extracted from livertissue, primary hepatocytes, or mouse hepa1c1c7 in lysis buffer (20 mMTris, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Trition X-100, 2.5mM β glycerol pyrophosphate, 1 mM NaVO₄, 1 mM NaF and 1 mM DTT.Immunoprecipitation of HA-TORC2 was performed according tomanufacturer's instructions.

DNA Plasmid Constructs:

Site-specific mutant TORC and CREB cDNAs were generated using theQuickchange protocol (Stratagene, La Jolla, Calif.) or by PCR-mediatedrestriction fragment recombination and then verified by sequencing.Protein expression of these constructs was evaluated by Western blot andimmunofluorescence analysis using anti-Flag or anti-GAL4 antibodies.TORC2 RNAi plasmid has been described (Conkright et al., 2003a).

SIK1 promoter sequences containing residues −425 to −76 were amplifiedby PCR from mouse genomic DNA and inserted into pXP2 Luc reporter vectorto genenate mSIK1 (−425/+76) Luc construct. PEPCK (−549/+49) Lucreporter, AOX Luc reporter, PGC-1 expression construct, and nonspecificcontrol pU6-US construct have been described previously (see Koo et al.,2004). pU6-RNAi plasmids were generated as described previously. Thecoding sequence from 468 to 488 (GGGGCAGTTGTTTAGACTGCC; SEQ ID NO:5) wasused for targeting mouse TORC2, and the coding sequence from 355 to 376(GGGCACTTGAGTGAAAACGAGG; SEQ ID NO:6) was used for targeting mouse/ratSIK1.

Transient Reporter Assays:

HEK293T, HIT or PC12 cells were co-transfected with EVX or GAL4-LUCreporter plasmid, RSV-beta galactosidase and FLAG-TORC and/or SIK2expression plasmids overnight using Lipofectamine 2000 (Invitrogen,Carlsbad, Calif.). DNA/lipid complexes were washed the next day andextracts prepared 24 h (293T and PC12) or 40 hr (HITs) aftertransfection. 45 mM KCl and/or 10 μM forskolin were added 6 hr prior toharvest and 7 hr hour after CsA treatment where indicated. Luciferasevalues were normalized to β-gal activity from co-transfected RSV-betagalactosidase activity in the corresponding sample.

In Vitro and In Vivo Phosphorylation Assays:

HIT and MIN6 insulinoma cells were transfected with Flag-tagged TORC1,TORC2, or TORC3 expression vector. In vivo: after 40 h, transfectedcells were incubated with phosphate-free Dulbecco's modified Eaglemedium containing 10% dialyzed serum and 1 mCi of[³²P]orthophosphate/ml. After 4 h, cells were washed with ice coldTris-buffered saline, and harvested in either boiling SDS or RIPA lysisbuffer (0.1% SDS, 1% Triton X-100, 0.5% deoxycholate, 50 mM Tris [pH8.0], 1 mM EDTA, 100 mM NaCl) containing a cocktail of phosphataseinhibitors (50 mM sodium fluoride, 1 mM sodium fluoride, 1 mM sodiumvanadate) plus 20 μg RNAse and 1 mM DTT. Samples were diluted to 1×RIPA(for boiling SDS samples) and immunoprecipitated after preclearing withStaph A using anti-Flag agarose (Sigma). In vitro: FLAG-TORC immunecomplexes were collected on Protein A-agarose, extensively washed andthen subjected to a kinase assay by incubation with 50 μCi γ-ATP and 10μM unlabeled ATP at 37° C. for 30 minutes in 50 mM HEPES, pH 7.4, 10 mMMgCl₂, 1 mM KCl. The reaction was washed once with reaction buffer andstopped by boiling in 2× Laemmli sample buffer.

Indirect Immunofluorescence:

Human ATYB1 fibroblasts on coverslips were fixed with 4% paraformadehydein PBS for 20 min. For staining of transient transfectants, cells werefixed 40 h after transfection. Fixative was quenched with 0.1M glycineprior to permeabilization with 0.1% TX-100/PBS for 2 min. Nonspecificsites were blocked with 3% BSA/PBS prior to staining with anti-FLAG M2monoclonal antibody (1:2000) or anti-pan TORC antiserum (1:1000) in 3%BSA/PBS. Antigens were revealed by incubation with donkey anti-mouse oranti-rabbit Alexa 488- or Cy3-conjugated secondary antibody (MolecularProbes, Eugene, Oreg., Jackson Immunochemicals) prior to mounting inVectashield with DAPI counterstain (Vector Labs). All steps wereperformed at 25° C.

Mass Spectrometry and Phosphorylation Site Determination:

Proteins resuspended in 8 M Urea, 100 mM Tris pH 8.5 were subjected toreduction and alkylation using 5 mM TCEP (Sigma) and 10 mM IAM (Sigma)respectively. Protein mixtures were then divided into three equalaliquots and diluted to 2 M Urea (4 M for Subtilisin) followed bydigestion with 0.01 μg/μl Trypsin (Promega), 0.005 μg/μl Elastase and0.005 μg/μl Subtilisin (Roche) (MacCoss et al., 2002). The resultingpeptide mixture was then analyzed by a 7 step MudPIT analysisessentially as described (Link et al., 1999), except proteins weredisplaced from the SCX to the RP column using the following salt stepgradients (1) 0% (2) 0-10% (3) 10-25% (4) 25-50% (5) 50-65% (6) 65-80%and (7) 80-100% of Buffer-A to Buffer-C. Peptides were eluted from theRP column into the mass spectrometer using a linear gradient of 15-55%of Buffer-A to Buffer-B. Mobile-phase buffers were, for Buffer-A, 95%H₂O, 5% acetonitrile, 0.1% formic acid; for Buffer-B, 20% H₂O, 80%acetonitrile, 0.1% formic acid; for Buffer-C, 500 mM NH₄OAc, 5%acetonitrile, and 0.1% formic acid. Tandem mass spectra were searchedagainst the most recent versions of the predicted rat, mouse, and humanproteins, to which common contaminants, such as keratin and trypsin,were added using a modified version of the PEP PROB algorithm. Searchresults were filtered and grouped using DTASelect (Tabb et al., 2002).For phosphorylation analysis a subset database was generated whichcontained only the proteins that were identified. The MSMS data werethen re-searched against this subset database for the modification of+80 on Ser/Thr/Tyr. The spectra containing the prominent 98-Da neutralloss were also searched against the subset database by using a modifiedversion of SEQUEST that considers the unique MS/MS fragmentationpatterns of phosphorylated Ser and Thr containing peptides (MacCoss etal., 2002). Only phosphorylation sites that were matched by multipletandem mass spectra representing sequences of different molecularweights (from the non-specific enzymes used in the digest) were calledmatches. Tandem mass spectra matched to phosphorylated peptides weremanually validated.

Culture of Primary Hepatocytes:

Rat primary hepatocytes were prepared from 200-300 g of Sprague-Dawleyrats by collagenase perfusion method as described previously (see Koo etal., 2004). 1×10⁶ cells were plated in 6 well plates with medium 199(Invitrogen, Carlsbad, Calif.) supplemented by 10% FBS, 10 units/mlpenicillin, 10 μg/ml streptomycin, and 10 nM dexamethasone for 3-6hours. After attachment, cells were infected with adenoviruses for 16hours. Subsequently, cells were maintained in medium 199 without FBS anddexamethasone for 24 hours (for cDNA expression adenoviruses) or 48hours (for RNAi adenoviruses) and 10 μM forskolin or 100 nM glucagon for2 hours unless noted otherwise. For experiments with cycloheximide, 10μg/ml cycloheximide was added 30 minutes prior to the addition of 100 nMglucagon.

Transfection Assays:

Human hepatoma HepG2 cells were maintained with Ham's F12 mediumsupplemented with 10% FBS (Invitrogen, Carlsbad, Calif.). Fortransfection, Fugene 6 reagent (Roche Applied Science) was usedaccording to the manufacture's instructions. Each transfection wasperformed with 100 ng of luciferase construct, 50 ng of betagalactosidase expression plasmid and 15 ng of expression vector forTORC2, 50 ng of expression vector for ACREB, 50 ng of expression vectorfor SIK1, or 50 ng of expression vector for PKA catalytic subunit. Ifnecessary, the empty pcDNA3 vector (Invitrogen, Carlsbad, Calif.) wasused for adding a constant amount for each transfection. 48 hours posttransfection, cells were treated with either DMSO or 10 μM Forskolin(Sigma, St. Louis, Mo.) for 4 hours and were harvested for luciferaseassays. The luciferase activity was normalized by beta galactosidaseactivity. For transfection with AOX reporter constructs, 100 ng ofluciferase construct, 50 ng of beta galactosidase expression plasmid and5 or 15 ng of expression vector for PGC-1α or 50 ng of expression vectorfor SIK1 were used.

Recombinant Adenoviruses:

Adenoviruses expressing GFP only and unspecific control were describedpreviously (see Koo et al., 2004). Adenoviruses for SIK1, SIK2, as wellas wild-type and Ser171Ala TORC2 expression were generated by homologousrecombination between adenovirus bacTORC2, SIK1 or SIK2 were generatedby homologous recombination between adenovirus backbone vector pAD-Easyand linearized transfer vector pAD-Track that contains mouse TORC2 cDNA,rat SIK1 cDNA or mouse SIK2 cDNA sequences, respectively. Adenovirusesfor TORC2 RNAi and SIK1 RNAi were generated by homologous recombinationbetween adenovirus backbone vector pAD-Easy and linearized transfervector pAD-Track that contains U6-TORC2 RNAi or U6-SIK1 RNAi sequences,respectively. The virus contained the cDNA that express GFP under thecontrol of CMV promoter for monitoring the infection efficiency. Foranimal experiments, viruses were purified by CsCl method and dialyzedagainst PBS buffer containing 10% glycerol before the injection.

Animal Experiments:

Male 7-week-old C57BL6 mice were purchased from Harlan and maintained inregular chow under the 12-h light-dark cycle. 0.5×10⁹ plaque-formingunits per recombinant adenovirus was delivered by a systemic tail veininjection to mice that were anaesthetized with Iso-Flurane. Formeasuring fasting blood glucose level, animals were fasted for 16 hourswith free access to water. Blood glucose was monitored at the end ofeach fasting period for 5 to 9 days. Liver tissues were collected at theend of experiments and immediately frozen in liquid nitrogen. Westernblots were performed with GFP antibody to check the relative infection.For the localization of TORC2 in liver, mice with adenovirus for mouseTORC2 were injected intraperitoneally with either 5 μg/kg body weight ofglucagon, 1 unit/kg body weight of insulin on PBS.

Metabolites:

Blood glucose level was monitored from tail vain blood using anautomatic glucose monitor (One Touch, Lifescan, Fremont, Calif.). Plasmainsulin levels were determined using a commercial insulin enzyme-linkedimmunosorbent assay kit (ALPCO Diagnostics, Windham, N.H.).

Immunostaining:

Formalin-fixed, paraffin-embedded liver sections (5 μm) weredeparaffinized in two changes of xylenes and hydrated to H₂O bysuccessive 5-min washes in 100% ethanol, 90% ethanol, 70% ethanol, anddistilled H₂O. Antigen unmasking was performed by microwaving slides for10 min in 1 mM EDTA. After cooling to room temperature, slides wererinsed twice in PBS. Slides were then incubated with PBS and 5% normaldonkey serum for 20 min. After incubation, slides were incubated withthe following rabbit antibodies for 60 mm at room temperature: antiTORC2(1:1600), anti-CREB (244, 1:600) and anti-pCREB (5322, 1:500). Afterantibody incubation and extensive washes, slides were incubated withdonkey anti-rabbit Cy3 at 1:600 dilutions for 45 mm at room temperature.Slides were then washed three times in PBS and mounted with coverslipswith the use of Vectashield mounting media containing 4′,6-diamidino-2-phenylindole (DAPI).

Chromatin IP:

Mice were injected intraperitoneally with either 5 μg/kg body weight ofglucagon or 1 unit/kg body weight of insulin for 10 minutes.Subsequently, nuclear isolation, chromatin crosslinking and ChiP assayswere performed as described (see Screaton et al., 2004). PrecipitatedDNA fragments were analyzed by quantitative polymerase chain reaction(Q-PCR) amplification using primers directed against the mouse promoterslisted in figure legends. For analysis of mouse SIK1 promoter,SV40-transformed mouse hepatocytes were gown to 90% confluence, and usedfor ChiP assays as described (see Screaton et al., 2004).SV40-transformed mouse hepatocytes were grown to 90% confluence, andChiP assays were performed. Precipitated DNA fragments were analyzed bypolymerase chain reaction (PCR) amplification using primers directedagainst the mouse SIK1 promoter or beta actin coding region as negativecontrol.

In Vitro Kinase Assay:

Recombinant GST-TORC2 (161-181) wild-type and Ser171Ala proteins werephosphorylated in vitro with 100 mU purified AMPK (AMP-activated proteinkinase) (Upstate Biotech)±300 μM AMP according to the manufacturer'sinstructions. Following 15 mm. incubation with γ-³²P-ATP, relative ³²Pincorporation was measured by scintillation. GST substrates and SAMSpeptide were used at 3 μM in each reaction.

Example 1 A Calcineurin Sensitive Cofactor Promotes CooperativityBetween cAMP and Calcium Pathways

Hamster insulinoma (HIT) cells were employed to test the relativeeffects of cAMP and calcium signals on CREB activity. Opening ofvoltage-sensitive L-type calcium channels in response to KCldepolarization induced the CREB-dependent EVX-1 promoter (Conknight etal., 2003b) 5-fold in HIT cells, whereas addition of cAMP agoniststimulated the reporter 10-fold (see FIG. 1A). Co-stimulation with bothforskolin and KCl stimulated CREB activity 80-fold, demonstrating thecooperative effects of cAMP and calcium pathways on CREB target geneexpression in these cells. Pre-treatment with the calcineurin inhibitorcyclosporine A (CsA) blocked cooperativity between cAMP and calciumpathways, indicating that calcincurin performs an important role inmodulating CREB activity. Consistent with the effects of calcium channelactivity on the EVX-1 promoter, treatment with high glucose (20 mM)induced the expression of the endogenous CREB target gene NR4A2 5-foldin MIN6 insulinoma cells; and co-stimulation with glucose plusexendin-4, an analogue of the incretin hormone GLP-1, enhanced NR4A2mRNA levels 35-fold (see FIG. 1B). In keeping with the importance ofcalcium entry for CREB target gene activation, treatment with thecalcium channel inhibitor nifedipine blocked the effect of glucose onNR4A2 mRNA accumulation. Cooperative induction of NR4A2 mRNA levels byglucose and exendin-4 was also calcincurin-dependent; addition of CsAdisrupted the increase in NR4A2 mRNA gene expression by both stimuli(see FIG. 1B).

The importance of Ser133 phosphorylation for CREB activation in responseto cAMP and calcium signals (Shaywitz and Greenberg, 1999) promptedexamination of the extent to which this site mediates cooperativitybetween both pathways. Treatment with forskolin and KCl increased levelsof CREB Ser133 phosphorylation in HIT cells (sec FIGS. 1C and 9); butco-stimulation with forskolin plus KCl had no additional effect onphospho (Ser 133) CREB levels relative to forskolin alone. Despite itsability to inhibit CREB activity in cells stimulated with cAMP andcalcium agonists, CsA had no effect either on the stoichiometry orkinetics of CREB Ser133 phosphorylation in response to these signals(see FIGS. 1C and 9). These results suggest that calcium and cAMP actsynergistically on a calcineurin regulated component which is distinctfrom the CREB Ser133 phospho-acceptor site.

The recruitment of CBP/P300 to the promoter is thought to be a commonpathway for activation of cellular genes via CREB in response to variousstimuli (Goodman and Smolik, 2000). To compare the effects of calciumand cAMP pathways in promoting the CREB:CBP interaction, mammaliantwo-hybrid assays were performed using GAL4-KID and KIX-VP16 expressionvectors. Addition of forskolin to HIT cells stimulated the KID:KIXinteraction 20-fold by GAL4 luciferase reporter assay; but KCldepolarization had no effect in this regard, despite its ability toinduce comparable levels of Ser133 phosphorylation (see FIG. 1D).Addition of forskolin with KCl rescued KID:KIX complex formation, albeitat similar levels to forskolin alone (see FIG. 1D). These resultsindicate that the cooperativity between calcium and cAMP signals on CREBtarget gene expression is not reflected at the level of the KID:KIXinteraction.

Previous reports suggesting that the CREB bZIP domain contributes totarget gene activation by CREB (Bonni et al., 1995a) prompted testing ofthe importance of this domain in mediating cooperativity between cAMPand calcium signals. In transient assays of HIT cells using a GAL4-CREBpolypeptide containing the GAL4 DNA binding domain fused to CREB, KCland forskolin induced GAL4 reporter activity 2 and 5-fold individuallybut 22-fold when added in combination; and these stimulatory effectswere potently inhibited by CsA (see FIG. 1E). By contrast, a truncatedGAL4-CREBΔbZIP polypeptide lacking the C-terminal bZIP domain (aa.284-341) in CREB showed only a modest response to forskolin and noinduction by KCl. Notably, the GAL4-CREBΔbZIP polypeptide elicited nocooperativity between cAMP and calcium agonists, revealing theimportance of the bZIP domain for this effect.

Example 2 TORCs Promote CREB Activation in Response to cAMP and CalciumSignals

The involvement of the CREB bZIP domain in mediating cooperativitybetween cAMP and calcium signals prompted examination of the role ofTORCs, a family of CREB coactivators which bind directly to the bZIP, inthis process. The TORC family comprises three members (TORC1, TORC2,TORC3) each of which contains highly conserved N-terminal CREB binding(aa. 1-42 in TORC1) and C-terminal trans-activation (aa. 517-634)domains (Conkright et al., 2003a; Iourgenko et al., 2003).Over-expression of TORC2 induced basal EVX-1 promoter activity 20-30fold; and treatment with cAMP or calcium channel agonist furtherpotentiated the reporter 25-fold and 30-fold, respectively, suggestingthat TORC2 is regulated by both signals (see FIG. 2A). Consistent withthe notion that TORC2 mediates CREB target gene expression via acalcineurin dependent mechanism, addition of CsA disrupted TORC2activity in HIT cells treated with KCl (see FIG. 2A).

To determine whether TORC2 is necessary for cAMP dependent induction ofCREB target genes, knockdown experiments were performed in HEK293T cellsfor their high transfection efficiency relative to HIT cells. Consistentwith its ability to potentiate CREB activity in response to cAMP andcalcium agonists, TORC2 was observed to facilitate target geneactivation; knockdown of TORC2 expression with a TORC2 RNAi constructreduced TORC2 protein levels and disrupted cAMP dependent induction ofthe EVX-1 promoter relative to a non-specific RNAi plasmid (Conkright etal., 2003a) (sec FIG. 2B, left panel). Expression of TORC3, which is notrecognized by the TORC2 RNAi construct, rescued induction of the EVX-1promoter by cAMP agonist (see FIG. 2B, right panel).

To further evaluate the role of TORCs in CREB activation, a screen wasconducted in efforts to identify TORC interaction-defective CREBpolypeptides. Based on previous data showing that the CREB bZIP domainmediates complex formation with TORC (Conkright et al., 2003 a), basicand leucine zipper regions were tested separately in pull down assays;it was determined that the leucine zipper motif alone was sufficient forbinding to TORC.

Mutagenesis studies on charged residues within the leucine zipper werethen carried out due to the sensitivity of the CREB:TORC complex to highsalt disruption. Out of five independent mutants tested, only one,Arg314Ala, showed substantially no TORC binding activity (see FIG. 10).Interestingly, Arg314 is conserved amongst CREB family members (CREB 1,ATF1, CREM), consistent with the suggestion of a potential role for thisresidue in CREB dependent transcription. By contrast with its potentactivity on wild-type CREB protein, TORC1 had no effect on the EVX-1reporter in cells expressing the CREB Arg314Ala mutant, demonstratingthe importance of this amino acid for CREB activation via TORC1 (seeFIG. 11).

To compare the relative contributions of CREB:CBP and CREB:TORCcomplexes for cellular gene induction, transient assays of HIT cellswere performed with GAL4-CREB polypeptides containing point mutationsthat disrupt interactions with CBP (M1: Ser133Ala), or TORC (M2:Arg314Ala). By contrast with the wild-type GAL4-CREB polypeptide, theCBP interaction defective M1 mutant was less responsive to cAMP (10-foldvs. 2.5 fold), but remained sensitive to cooperative effects of cAMP andKCl as well as to the inhibitory effects of CsA (see FIG. 2C).Mutagenesis of Arg314 in GAL4-CREB (M2) not only disrupted cAMPinducibility, but also compromised cooperativity between cAMP and KCl(see FIG. 2C). Similar results were noted in other electricallyexcitable cells including PC12 pheochromocytoma cells (see FIG. 12),consistent with the hypothesis that TORCs function in a variety ofcellular contexts.

The apparent requirement of CBP and TORC complexes for cellular geneactivation via CREB prompted examination of whether these proteins arerecruited to the promoter by chromatin immunoprecipitation (ChIP) assay.This analysis was conducted in HEK293T cells due to the absence ofhamster genomic sequence data for CREB target genes. In ChIP assays onthe endogenous CREB target gene NR4A2 (Conkright et al., 2003b), it wasfound that CREB occupied the promoter both under basal conditions and inresponse to cAMP treatment (see FIG. 2D). CBP and TORC2 were absent fromthe NR4A2 promoter under basal conditions; following treatment withforskolin for 30 minutes, both were recruited to the promoter but not toa control 3′ end fragment of the NR4A2 gene. These results areconsistent with functional data from both TORC2 knockdown and CREBmutagenesis assays (see FIGS. 2B and 2C) demonstrating the role of TORC2as a signal-dependent CREB coactivator.

Example 3 TORCs Migrate to the Nucleus in Response to cAMP

Immunofluorescence studies were performed to determine the mechanism bywhich cAMP triggers recruitment of TORC2 to the promoter. The relativeabsence of cytoplasm in HIT cells for microscopic analysis of TORClocalization prompted the use of human ATYB1 fibroblasts. Flag-taggedand endogenous TORC2 proteins were largely confined to the cytoplasm ofATYB1 cells under basal conditions; and treatment with cAMP agonistpromoted nuclear accumulation of TORC2 within 30 minutes (see FIGS. 3Aand 13). Treatment with the exportin inhibitor leptomycin B (LMB)strongly enhanced nuclear accumulation of both endogenous andtransfected TORC1 and TORC2 within 30 to 60 minutes, indicating thatthese proteins likely cycle in and out of the nucleus in the absence ofcellular stimulus (see FIGS. 3B and 13).

To characterize regions in TORC that promote either cytoplasmic ornuclear accumulation, cellular fluorescence assays were performed ontruncated TORC1 polypeptides fused to green fluorescent protein (GFP).Nuclear localizing activities (NLS1 and NLS2) were detected near theN-terminus of TORC1 (aa. 1-147), whereas nuclear export sequences (NES1, NES2) were located within the central Ser/Pro rich domain from aa.148-290 (see FIG. 14). Both NLS and NES motifs were well conservedwithin the three TORC family members, and mutagenesis of individualleucines in NES 1 and NES2 promoted nuclear accumulation of TORCs.

TORC3 contains a Phe to Tyr282 substitution in NES 1, which would bepredicted to disrupt export activity (see FIG. 3C, bottom). Indeed,TORC3 was localized exclusively in the nucleus of both control and cAMPstimulated cells (see FIG. 3C, top); consistent with its nuclearlocation, wild-type TORC3 was far more active in potentiating CREBactivity under basal conditions relative to TORC2 (see FIG. 3D).Conversely, addition of an N-terminal src myristylation signal targetedTORC3 to the cytoplasm and rendered the protein transcriptionallyinactive (see FIG. 3D). Likewise, mutagenesis of Tyr282 in TORC3 to Phepromoted cytoplasmic accumulation of the protein and repressed basalTORC3 activity. cAMP treatment triggered translocation of mutant TORC3Tyr282Phe to the nucleus, consistent with the notion that cAMPstimulates nuclear entry of TORC proteins (see FIG. 3C).

Example 4 TORC is Dephosphorylated in Response to cAMP and CalciumSignals

To determine the mechanism by which extracellular signals regulate TORC2nuclear entry, the biochemical properties of TORC2 were compared innuclear and cytoplasmic fractions of HEK293T cells. Flag-tagged TORC2appeared as an 85 kD doublet in cytoplasmic fractions and as a singlefaster migrating species in nuclear extracts (see FIG. 4A, left panel).Indeed, treatment with calf intestinal alkaline phosphatase (CIP)transformed the cytoplasmic TORC2 doublet into a single faster migratingspecies, consistent with the belief that TORC2 undergoesdephosphorylation upon nuclear entry (see FIG. 4A, right panel).

To determine whether TORC2 phosphorylation is regulated in response tocalcium and cAMP signals, Western blot assays were performed onendogenous TORC2 protein in HIT cells. Consistent with results using theFlag-tagged protein, endogenous TORC2 also appeared as two closelymigrating bands in total extracts (see FIG. 4B). Co-stimulation with KClplus forskolin promoted extensive TORC2 dephosphorylation; andpre-treatment with CsA partially reversed this effect, consistent withthe belief that calcineurin promotes TORC2 dephosphorylation in responseto these inducers (see FIG. 4B).

To evaluate the status of TORC2 phosphorylation directly, ³²P-labelingexperiments were performed. SDS-PAGE analysis of flag-tagged TORC2immunoprecipitates from HIT cells revealed that in vivo ³²P-labeledTORC2 was dephosphorylated within 10 minutes after treatment withforskolin or KCl (see FIG. 4C). Co-stimulation with forskolin and KClreduced phospho-TORC2 levels further; these effects were blocked byco-treatment with CsA, consistent with the belief that calcineurinpromotes TORC2 dephosphorylation in this setting (see FIG. 4C). TORC2appears to be phosphorylated exclusively on serine by phospho-amino acidanalysis; and two dimensional tryptic mapping of ³²P-labeled TORC2reveals at least seven spots of comparable intensity, indicating thatTORC2 is extensively phosphorylated at numerous sites.

Example 5 TORCs Associate with 14-3-3 Proteins

To clarify the mechanism by which TORC phosphorylation promotes itscytoplasmic retention, proteomic analyses were performed to search forTORC interacting proteins. Immunoprecipitates of TORC1 and TORC2 wereprepared from stable cell lines expressing Flag-tagged versions ofeither protein. Both TORCs were found to interact strongly with multiplemembers of the 14-3-3 family of proteins (e.g., 92.5% coverage for14-3-3ε). 14-3-3 proteins have been found to bind a number of regulatoryproteins, most notably CDC25A, forkhead, and NFAT family members, and toinhibit their biological function (Brunet et al., 1999; Chen et al.,2003; Chow and Davis, 2000; Durocher et al., 2000).

Co-immunoprecipitation studies were performed to confirm the proteomicresults and to explore the potential role of 14-3-3 proteins inregulating TORC activity. Endogenous 14-3-3 proteins were recovered fromimmunoprecipitates of Flag-TORC1 and Flag-TORC2 expressing cells as wellas from immunoprecipitates of endogenous TORC proteins from HEK293T andPC12 cells (see FIG. 5A). Consistent with its ability to promote TORCtranslocation to the nucleus, forskolin treatment disrupted bothtransfected Flag-TORC1:14-3-3 (see FIG. 5A, top panel) and endogenousTORC:14-3-3 interactions (see FIG. 5A, bottom left panel). The kineticsof TORC2:14-3-3 dissociation parallel the time course for TORC2dephosphorylation and nuclear entry in response to cAMP agonist; inco-immunoprecipitation studies of Flag-TORC2 and 14-3-3 proteins, theTORC2:14-3-3 interaction was diminished by about half within 10 minutesof forskolin treatment and was maximally reduced after 30 to 60 minutes(see FIG. 5A, bottom right panel).

The general importance of Ser/Thr phosphorylation for association with14-3-3 family members prompted examination of whether the inhibitoryeffects of CsA on TORC activity correlate with changes in TORC: 14-3-3binding. CsA treatment greatly enhanced complex formation under basaland cAMP stimulated conditions, whereas the protein phosphatase PP1/PP2Ainhibitor okadaic acid (OA) had no effect on this interaction. Thesedata are consistent with the belief that calcineurin mediatesdissociation of TORC:14-3-3 complexes in response to calcium signals bydephosphorylation of TORCs (see FIG. 5A).

Co-immunoprecipitation studies were performed on mutant TORCpolypeptides to identify regions in TORC2 that mediate the 14-3-3interaction. The central Ser/Pro rich domain in TORC2 (aa. 56-547)appeared important in this regard; relative to other mutant TORC2polypeptides, no endogenous 14-3-3 proteins were recovered fromimmunoprecipitates of mutant TORC2 (Δ56-547) (see FIG. 5B). Consistentwith its proposed role in promoting cytoplasmic retention of TORCproteins, over-expressed 14-3-3 beta inhibited basal EVX-1 reporteractivity in HEK293T cells transfected with a TORC2 expression vector. Bycontrast, 14-3-3 beta over-expression had no effect on the activity ofthe TORC2 (Δ56-547) protein, demonstrating the importance of the TORC:14-3-3 interaction for repression of CREB target genes (see FIG. 5C).

The ability of CsA to block TORC2 dephosphorylation and to enhance the14-3-3 interaction prompted an evaluation of the role of calcineurin inthis process. Remarkably, calcineurin A and B subunits were identifiedin proteomic analyses of Flag-tagged TORC1 (10.4% peptide coverage) andTORC2 (7.9% peptide coverage) immunoprecipitates. TORC2 appears to bindto calcineurin A directly; in pull-down assays ³⁵5-labeled TORC2 wasefficiently precipitated with GST-calcineurin A (aa. 1-347) but not GSTbeads (see FIG. 5D, bottom panel). The 14-3-3 interaction-defectiveTORC2 (Δ56-547) protein, lacking the central regulatory domain, was alsounable to associate with calcineurin, demonstrating the importance ofthis region in TORC2 for signal dependent modulation.

Example 6 TORC2 Associates with SIK2, a Snfl Related Kinase

The high levels of cytoplasmic TORC2 phosphorylation on serine underbasal conditions prompted testing to determine whether TORCs associatewith a Ser/Thr kinase activity. Both endogenous and over-expressed TORC2were readily phosphorylated by in vitro kinase assay ofimmunoprecipitates prepared from cytoplasmic (C) but not from nuclear(N) fractions of HEK293T cells (see FIG. 6A). TORC associated kinaseactivity was potently inhibited by treatment with forskolin (compareintensities of 85kD TORC2 bands), consistent with one of twoexplanations, i.e., that PKA either reduces the activity of the kinaseor disrupts the TORC2:kinase complex. Two dimensional phospho-trypticmapping studies of ³²P-labeled flag-tagged TORC2 revealed a single majorspot following in vitro kinase assay of TORC2 immunoprecipitates,consistent with the explanation that TORC associated kinasephosphorylates TORC2 at one principal site rather than at the multiplesites observed in vivo.

Proteomic analysis of Flag-TORC2 immunoprecipitates prepared fromtransfected HEK293T cells revealed the presence of the Salt InducibleKinase 2 (SIK2: 6 peptides, 11.7% coverage), a member of the snf1 familyof energy-sensing kinases previously found to inhibit transcription ofcAMP responsive genes (Doi et al., 2002). Confirming this finding, SIK2was readily detected in anti-flag immunoprecipitates prepared from11EK293T cells co-transfected with SIK2 and Flag-tagged TORC2 expressionvectors (see FIG. 6B). Moreover, TORC2 mobility was noticeably reducedin cells co-expressing SIK2, consistent with the suggestion that thiskinase directly phosphorylates TORC2 (see FIG. 6B, compare lanes 3,4).

To identify sites on TORC2 that are phosphorylated by SIK2 and othercellular kinases, tandem mass spectroscopic (MS/MS) analysis wasperformed of TORC2 phospho-peptides generated from TORC2immunoprecipitates. Eleven phospho-peptides were recovered by thisanalysis; and most of these mapped to the central regulatory domain (seeFIG. 6C SEQ ID NOS: 17-27). Remarkably, one TORC2 phospho-peptidecorresponded to an optimal site for SIK2 phosphorylation (LXB(S/T)XSXXXL(SEQ ID NO:7): aa.166-LNRTSSDSAL (SEQ ID NO:8) in TORC2 (see FIG. 15-SEQID NOS: 1, 28-53)). To determine whether Ser171 in TORC2 is indeedphosphorylated by SIK2 in vitro kinase assays were performed using aGST-TORC2 (aa 161-181) substrate. In ³²P-labeling studies, SIK2 wasfound to phosphorylate wild-type but not Ser171Ala mutant TORC2 invitro, consistent with the suggestion that SIK2 phosphorylates TORC2 ata single site under basal conditions (sec FIG. 6D).

Example 7 SIK2 Inhibits TORC2 Nuclear Translocation

cAMP has been reported to disrupt the inhibitory effects of SIK2 on CREBactivity via the PKA mediated phosphorylation of SIK2 at Ser587 (Okamotoet al., 2004). Indeed, treatment with cAMP agonist inducedphosphorylation of SIK2 at Ser587, by Western blot assay of GST-SIK2expressing cells with phospho-Ser(587) specific antiserum (see FIG. 7A).Consistent with its proposed role in regulating CREB activity,endogenous SIK2 was readily detected in HIT cell extracts by Westernblot assay. To further explore the functional role of SIK2 in modulatingCREB-dependent gene expression, transient assays were performed onHEK293T cells co-transfected with the EVX-1 reporter plasmid.Over-expression of wild-type SIK2 blocked reporter activity about 70% incAMP stimulated cells; but kinase dead SIK2 (K49M) had no effect in thisregard, demonstrating the importance of SIK2 catalytic activity for CREBinhibition (see FIG. 7B, left panel). PKA phosphorylation defective SIK2(Ser587Ala) was far more potent in reducing CRE reporter activity,consistent with the belief that cAMP normally disrupts SIK2 activity inthis context (see FIG. 7B, left panel).

To evaluate the effect of Ser171 phosphorylation by SIK2 on TORC2activity, transient assays were performed on a Ser171Ala mutant TORC2expression vector. Relative to the wild-type protein, TORC2 (Ser171Ala)was far more active in potentiating CREB activity under basalconditions, but displayed comparable activity to wild-type TORC2following forskolin treatment (see FIG. 7B, right panel). These resultsare consistent with the suggestion that SIK2 dependent phosphorylationat Ser171 represses TORC2 under basal conditions; and that cAMPstimulates TORC2 activity by disrupting SIK2 mediated Ser 171phosphorylation.

The importance of TORC2 nuclear entry for target gene activation inresponse to cAMP prompted examination of the effect of SIK2 on TORC2localization. Under basal conditions, flag-tagged TORC2 was localized toboth nuclear and cytoplasmic compartments of ATYB1 cells (see FIG. 7C).Over-expression of SIK2 efficiently blocked nuclear entry of TORC2 underbasal conditions; and treatment with forskolin promoted nuclear entry ofTORC2 in these cells, demonstrating the ability of PKA to overcome theinhibitory effects of SIK2 on TORC2 translocation. By contrast, TORC2remained cytoplasmic even following cAMP treatment in cells expressingthe PKA phosphorylation defective SIK2 (Ser587Ala), reinforcing the roleof Ser587 for TORC2 activation. Phosphorylation of Ser171 by SIK2appears important for cytoplasmic retention; mutant TORC2 (Scr171Ala)was targeted to the nucleus constitutively under both basal and cAMPstimulated conditions (see FIG. 7C). Taken together, these results areconsistent with the proposal that the SIK2 mediated phosphorylation ofTORC2 at a single site (Ser171) favors cytoplasmic retention of TORC2and inhibition of CREB activity under basal conditions.

Example 8 TORC2, a Master Switch for Hepatic Gluconeogenesis

Under fasting conditions, pancreatic glucagon triggers the activation ofcatabolic programs in liver in part via the cAMP responsive factor CREB(see Herzig et al., 2001; Hall & Granner, 1999; and Hanson & Reshef,1997). CREB in turn stimulates gluconeogenesis and fatty acid oxidationgenes by inducing expression of the nuclear hormone receptor coactivatorPGC-1α (see Herzig et al., 2001 and Voon et al., 2001). Consistent withthe above, mice deficient in PGC-1α display defects in hepaticgluconeogenesis and fatty acid oxidation (see Koo et al., 2004 and Linet al., 2004).

Glucagon is thought to enhance CREB activity via the PKA mediatedphosphorylation of CREB at Ser133, and this modification in turnstimulates target gene expression via the recruitment of the coactivatorCBP to the promoter (see Chrivia et al., 1993 and Arias et al., 1994).Intraperitoneal (IP) administration of glucagon was found to promoteCREB Ser133 phosphorylation in liver within 10 minutes by histochemicaland Western blot analysis (see FIGS. 16A and 16B). Unexpectedly, IPinsulin administration had comparable effects on Ser133 phosphorylationin the liver, arguing against a pivotal role for the CREB:CBP pathway indiscriminating between fasting and feeding signals (see FIGS. 16A and16B).

In addition to promoting CREB phosphorylation, cAMP has also been foundto stimulate cellular gene expression via the dephosphorylation andnuclear entry of TORCs, a family of cytoplasmic coactivators thatenhances cellular gene expression via an interaction with the CREB basicregion/leucine zipper (bZIP) DNA binding domain (see Conkright et al.,2003a and Iourgenko et al., 2003). Thus, TORC2 activity was examined, asthis family member was expressed at highest levels relative to TORC1 andTORC3 in liver as determined by quantitative PCR analysis. Hepatic TORC2was localized primarily in the cytoplasm under ad libitum feedingconditions by immuno-histochemical analysis of liver sections (see FIG.16C). IP glucagon administration induced translocation of TORC2 to livernuclei within 10 minutes. Despite its ability to promote CREB Ser133phosphorylation, insulin did not stimulate nuclear entry of TORC2,demonstrating the capacity of this coactivator to discriminate betweenfasting and feeding signals. Consistent with these dynamics, TORC2 washighly phosphorylated at Ser171 under ad libitum or insulin stimulatedconditions, but was dephosphorylated following glucagon induction (seeFIG. 16D). Moreover, in chromatin immunoprecipitation (ChIP) assays ofliver tissue, glucagons, but not insulin, promoted recruitment of TORC2to gluconeogenic genes (sec FIG. 16E). Taken together, these resultsdemonstrate that, by contrast with CREB Ser 133 phosphorylation, TORC2activity is selectively induced in response to fasting signals.

Based on its ability to translocate to the nucleus in response toglucagon, TORC2 would be expected to enhance gluconeogenic geneexpression in a cAMP regulated manner. This proposal was tested byinfecting primary rat hepatocytes with a TORC2 expressing adenovirus(Ad-TORC2). Ad-TORC2 had marginal effects on gluconeogenic genes(PGC-1α, PEPCK, and glucose 6 phosphatase) under basal conditions butpotentiated the entire program 10-fold following exposure to Forskolin(FSK) (see FIG. 17A). The effects of Ad-TORC2 on gluconeogenic geneexpression were CREB dependent; expression of a dominant negative A-CREBpolypeptide, which specifically inhibits binding of CREB but not otherbZIP family members to DNA (see Ahn et al., 1998), disrupted Ad-TORC2potentiation (see FIG. 17B).

The ability of TORC2 to modulate gluconeogenic gene expression inhepatocytes exposed to cAMP agonist is consistent with the proposal thatthis coactivator modulates glucose output from the liver in response tofasting signals. Expression of either Ad-TORC2 or its paralog TORC1 inprimary rat hepatocytes enhanced glucose output nearly 5-fold in cellsexposed to FSK plus dexamethasone (see FIG. 17C); and glucose outputfrom TORC over-expressing cells was disrupted by insulin treatment,demonstrating the ability of this coactivator to respond appropriatelyto both fasting and feeding signals. When expressed at levels comparableto the endogenous protein in liver, Ad-TORC2 promoted fastinghyperglycemia (see FIG. 17D). Levels of circulating insulin wereelevated commensurately in Ad-TORC2 mice, indicating that the effects ofthis coactivator on hepatic glucose output are sufficient to trigger acounter-regulatory response.

Based on its ability to stimulate the gluconeogenic program and topromote hyperglycemia when over-expressed in mice, endogenous TORC2 isexpected to regulate the response to fasting signals in liver. Thisexpectation was tested employing a TORC2 RNAi construct that reducedexpression of TORC2 proteins nearly 80% by Western blot assay (see FIG.17E). Mice made acutely deficient in TORC2 by injection with the TORC2RNAi adenovirus exhibited fasting hypoglycemia (60 mg/dl vs 100 mg/dl;see FIG. 17E); and mRNAs for gluconeogenic genes were reduced 3-fold onaverage relative to control littermates (see FIG. 17F).

Like other signaling pathways, cAMP stimulates gluconeogenic geneexpression with burst-attenuation kinetics (see Sasaki et al 1984).Following exposure of primary hepatocytes to Forskolin, PEPCK mRNAlevels became maximal after 2 hours, returning to near baseline after 4hours (see FIG. 18A). Consistent with this profile, TORC2phosphorylation at Ser171 was strongly induced 3 hours after glucagonstimulation in primary rat hepatocytes (see FIG. 18B). Pre-treatmentwith protein synthesis inhibitor cycloheximide (CHX) blockedphosphorylation of TORC2 at Ser171 by glucagon at 3 hours, suggestingthat fasting signals promote the synthesis of an activity which in turnfeeds back to shut down the CREB:TORC pathway (see FIG. 18B). Consistentwith this, CHX pre-treatment potentiated gluconeogenic gene expressionin primary rat hepatocytes exposed to glucagon 2-3 fold (see FIG. 18C).

Based on the ability of CHX to disrupt TORC2 Ser171 phosphorylation, itwas hypothesized that glucagon induces the expression of an inhibitorykinase during the attenuation period. In previous studies, the SIKfamily of AMP kinases has been found to associate with and tophosphorylate TORC2 at Ser171 (sec Screaton et al., 2004), part of anoptimal consensus site for AMPK (AMP-activated protein kinase) familymembers (LNRTSSDSAL; SEQ ID NO:9). Indeed, fasting induced SIK1 mRNA andprotein levels in liver 4-fold relative to feeding conditions, whereasexpression of other SIK family members (SIK2 and SIK3) was unaffected(see FIGS. 18D and 18E). Exposure of primary rat hepatocytes to FSKstrongly induced SIK1 mRNA levels 20-fold, and these effects weredisrupted by Ad-A-CREB (see FIG. 18F). Notably, FSK had no effect onmRNA levels for any of the 12 AMPK family members by gene profilingassay of primary mouse hepatocytes, indicating that these effects areindeed specific for SIK1.

Examination of the SIK1 gene promoter revealed two consensus cAMPresponsive promoter elements in rat, mouse, and human orthologs,consistent with the proposal that SIK1 is a direct target for CREBinduction. In transient assays of HepG2 hepatocytes, PKA stimulated SIK1promoter activity about 20-fold; these effects were disrupted byco-expression of A-CREB (see FIG. 18G). Indeed, CREB was found to occupythe SIK1 promoter in ChIP assays of mouse hepatocytes, consistent withthe proposed direct role for CREB in this process (see FIG. 18H).

Having seen that glucagon triggers hepatic expression of SIK1 duringfasting, it was next considered whether this kinase functions as part ofan auto-regulatory loop in attenuating the gluconeogenic program. Usinga SIK1 RNAi adenovirus that reduced SIK1 expression about 75%, it wasfound that glucagon stimulated TORC2 dephosphonylation to a far greaterextent in SIK1 knockdown compared to control cells (see FIG. 19A).Knockdown of SIK1 also enhanced gluconeogenic gene expression; mRNAlevels for PGC-1α were increased 70-fold in SIK1 deficient cells (seeFIG. 19B). Conversely, over-expression of SIK1 induced Ser171phosphorylation and blocked induction of the PEPCK promoter by TORC2 inhepatocytes exposed to FSK. However, SIK1 had no effect on the abilityof PGC-1 to stimulate transcription from a PPARα target gene (Acyl CoAoxidase (AOX)), which is not consistent with the existence of a generalinhibitory effect of this kinase on hepatocyte gene expression.

The role of SIK1 on fasting glucose metabolism was further evaluatedusing Ad-SIK1. Relative to control littermates, mice injected witheither Ad-SIK1 or Ad-SIK2 exhibited fasting hypoglycemia and reducedgluconeogenic gene expression (see FIGS. 19C and 19D). Although theability of Ad-SIK1 to block hepatic glucose output in this setting couldreflect an unanticipated induction of the insulin pathway, fastinginsulin levels were actually lower in SIK1 or SIK2 expressing mice,compared to control mice; hepatic insulin signaling was comparable inprimary hepatocytes infected with a SIK1 adenovinus, as revealed byWestern blot assay of phospho (Ser473) Akt levels following 30 minexposure to insulin (100 nM).

To evaluate whether SIK1 inhibits the gluconeogenic program via TORC2phosphorylation, an adenovinus expressing Ser171Ala TORC2 was prepared.Following infection into primary rat hepatocytes, adenoviral wild-typeand mutant Ser171Ala TORC2 polypeptides were expressed at comparablelevels and had similar effects on PEPCK and PGC-1α gene expression (seeFIGS. 19E and 19F). Over-expression of SIK1 blocked wild-type TORC2activity almost completely; but the mutant Ser171Ala TORC2 protein wasrefractory to SIK1 inhibition, demonstrating the importance of Ser171phosphorylation for disruption of the gluconeogenic program by thiskinase (see FIGS. 19E and 19F).

Activation of the AMPK (AMP-activated protein kinase) pathway in liverhas been shown to block expression of gluconeogenic genes, although therelevant targets for this inhibition have remained elusive (see Lockheadet al., 2000 and Yamauchi et al., 2002). By contrast with SIK, ATPdepletion appears to be uniquely sensed by AMPK, the founding member ofthis family (see Sakamoto et al., 2004; Lizcano et al., 2004; Bananjeeet al., 2004; and Radzuik et al., 2003). The presence of a consensusAMPK phosphorylation site (Ser171) that modulates TORC2 activity inliver prompted examination of whether AMPK inhibits gluconeogenesis viaphosphorylation of TORC2 in response to ATP depletion. Activated AMPKphosphorylated wild-type, but not Ser171Ala mutant GST-TORC2 (161-181)peptide in vitro (see FIG. 20A). Phosphorylation of TORC by AMPK wascomparable to an optimal AMPK peptide substrate (SAMS (see Lizcano etal., 2004) and was induced by addition of AMP. Indeed, selectiveactivation of cellular AMPK by exposure of primary hepatocytes to theAMP analogue 5-aminoimidazole-4-carboxamide riboside (AICAR) triggeredrobust phosphorylation of endogenous TORC2 even in the presence of FSK(see FIG. 20B).

Based on its ability to promote Ser171 phosphorylation, AMPK might beexpected to inhibit nuclear entry of TORC2 in cells exposed to FSK.Using primary rat hepatocytes infected with adenoviruses expressingeither wild-type or Ser171Ala mutant TORC2 polypeptides, it was foundthat FSK triggered translocation of wild-type TORC2 (see FIG. 20C).Confirming the pivotal role of Ser171 in this regard, mutant Ser171AlaTORC2 remained constitutively nuclear under both conditions. Treatmentwith AICAR inhibited nuclear entry of wild-type, but not SerAla171TORC2, in cells exposed to FSK; and adenoviral expression of SIK1 inthese cells similarly blocked TORC2 nuclear entry in a Ser171 dependentmanner (see FIG. 20C).

Activation of AMPK (AMP-activated protein kinase) by AICAR in primaryhepatocytes to AICAR blocked induction of PEPCK and PGC-1α genes inresponse to FSK (see FIG. 20D). If the AMPK pathway inhibits thegluconeogenic program via TORC2, then a phosphorylation defectiveSer171Ala mutant TORC2 would be predicted to rescue the inhibitoryeffects of AICAR on these genes. Consistent with this prediction, mutantAd-TORC (Ser171Ala) rescued expression of PEPCK and PGC-1α in thepresence of AICAR inhibitor, demonstrating the importance of Ser171 inmediating inhibitory effects of AMPK on gluconeogenic genes (see FIG.20D).

The results described herein are consistent with a mechanism of actionwhereby TORC2 functions as a master switch for modulation ofgluconeogenic genes in response to nutritional and stress signals (seeFIG. 20E). TORC2 activity is tightly regulated during fasting by SIK1,which forms part of an autoregulatory loop that attenuates thegluconeogenic program by phosphorylating TORC2 at Ser171 and promotingits export to the cytoplasm. TORC2 is additionally regulated by AMPK;activation of AMPK in response to an AMP analog disrupted hepaticgluconeogenic gene expression in part via Ser171 phosphorylation ofTORC2. Indeed, a number of adipokines such as adiponectin (see Yamauchiet al., 2002) and resistin (sec Bananjee et al., 2004) have been shownto modulate hepatic gluconeogenesis via an AMPK dependent mechanism; theresults presented herein provide a regulatory mechanism to explain theseeffects. For example, metformin, a compound that activates AMPK (seeRadzuik et al., 2003), has been widely used for treatment of type IIdiabetes due to its effects in blocking hepatic gluconeogenesis and instimulating glucose uptake in muscle (see also Bergeron et al., 2001).Other compounds that enhance TORC2 phosphorylation in liver would beexpected to provide similar therapeutic benefit for individuals withinsulin resistance.

The invention has been described in detail with reference to preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may make modificationand improvements within the spirit and scope of the invention as setforth in the following claims.

All publications, patent applications, issued patents, and otherdocuments referred to in this specification are herein incorporated byreference as if each individual publication, patent application, issuedpatent, or other document was specifically and individually indicated tobe incorporated by reference in its entirety. Definitions that arecontained in text incorporated by reference are excluded to the extentthat they contradict definitions in this disclosure.

REFERENCES

-   Ahn. S. et al. A dominant negative inhibitor of CREB reveals that it    is a general mediator stimulus-dependent transcription of c-fos.    Molec. Cell. Biol. 18, 967-977 (1998).-   Al-Uzri, A., Stablein, D. M., and Cohn, A. R. (2001). Posttransplant    diabetes mellitus in pediatric renal transplant recipients: a report    of the North American Pediatric Renal Transplant Cooperative Study    (NAPRTCS). Transplantation 72, 1020-1024.-   Aramburu, J., Garcia-Cozar, F., Raghavan, A., Okamura, H., Rao, A.,    and Hogan, P. G. (1998). Selective inhibition of NFAT activation by    a peptide spanning the calcineurin targeting site of NFAT. Mol Cell    1, 627-637.-   Arias, J., Alberts, A., Brindle, P., Claret, F., Smeal, T., Karin.    M., Feramisco, J., and Montminy, M. (1994). Activation of CAMP and    mitogen responsive genes relies on a common nuclear factor. Nature    370, 226-228.-   Asahara, H., Santoso, B., Du, K., Cole, P., and Montminy, M. (2001).    Chromatin Dependent Cooperativity Between Constitutive and Inducible    Activation Domains in CREB. Molecular and Cellular Biology 21,    7892-7900.-   Banenjee, R. R. et al. Regulation of fasted blood glucose by    resistin. Science 303, 1195-8 (2004).-   Bergeron, R. et al. Effect of    5-aminoimidazole-4-canboxamide-1-beta-Dribofuranoside infusion on in    vivo glucose and lipid metabolism in lean and obese Zucker rats.    Diabetes 50, 1076-82 (2001).-   Bittinger, M. A. et al. Activation of cAMP response element-mediated    gene expression by regulated nuclear transport of TORC proteins.    Curr Biol 14, 2156-61(2004).-   Bonni, A., Ginty, D., Dudek, H., and Greenberg, M. (1995a). Serine    133 phosphorylated CREB Induces Transcription via a Cooperative    Mechanism That May confer Specificity to Neurotrophin Signals.    Molecular and Cellular Neurosciences 6, 168-183.-   Bonni, A., Ginty, D. D., Dudek, H., and Greenberg, M. E. (1995b).    Serine 133-phosphorylated CREB induces transcription via a    cooperative mechanism that may confer specificity to neurotrophin    signals. Mol Cell Neurosci 6, 168-183.-   Brunet, A., Bonni, A., Zigmond, M. J., Lin. M. Z., Juo, P., Hu, L.    S., Anderson, M. J., Arden, K. C., Blenis, J., and Greenberg, M. E.    (1999). Akt promotes cell survival by phosphorylating and inhibiting    a Forkhead transcription factor. Cell 96, 857-868.-   Carling, D. (2004). The AMP-activated protein kinase cascade—a    unifying system for energy control. Trends Biochem Sci 29, 18-24.-   Chen, M. S., Ryan, C. E., and Piwnica-Worms, H. (2003). Chk1 kinase    negatively regulates mitotic function of Cdc25A phosphatase through    14-3-3 binding. Mol Cell Biol 23, 7488-7497.-   Chow, C. W., and Davis, R. J. (2000). Integration of calcium and    cyclic AMP signaling pathways by 14-3-3. Mol Cell Bio 120, 702-712.-   Chrivia, J. C., Kwok, R. P., Lamb, N., Hagiwara, M., Montminy. M.    R., and Goodman, R. H. (1993). Phosphorylated CREB binds    specifically to the nuclear protein CBP. Nature 365, 855-859.-   Conkright, M. D., Canettieri, G., Screaton, R., Guzman, E.,    Miraglia, L., Hogenesch, J. B., and Montminy, M. (2003a). TORCs:    transducers of regulated CREB activity. Mol Cell 12, 413-423.-   Conkright, M. D., Guzman, E., Flechner, L., Su, A. I., Hogenesch,    J., and Montminy, M. (2003b). Genome Wide Analysis of CREB Target    Genes Reveals A Core Promoter Requirement for CAMP Responsiveness.    Mol Cell 11, 1101-1 108.-   Crabtree, G. R., and Olson, E. N. (2002). NFAT signaling:    choreographing the social lives of cells. Cell 109 Suppl, S67-79.-   Doi, J., Takemori, H., Lin, X. Z., Horike, N., Katoh, Y., and    Okamoto, M. (2002). Saltinducible kinase represses CAMP-dependent    protein kinase-mediated activation of human cholesterol side chain    cleavage cytochrome P450 promoter through the CREB basic leucine    zipper domain. J Biol Chem 277, 15629-15637.-   Dougherty, M. and Morrison, D. (2004). Unlocking the code of 14-3-3.    J Cell Science 117:1875-1884.-   Durocher, D., Taylor, I. A., Sarbassova, D., Haire, L. F.,    Westcon, S. L., Jackson, S. P., Smerdon, S. J., and Yaffe, M. B.    (2000). The molecular basis of FHA domain:phosphopeptide binding    specificity and implications for phospho-dependent signaling    mechanisms. Mol Cell 6, 1169-1 182.-   Enlund, F., Behhoudi, A., Andren, Y., Oberg, C., Lendahl, U., Mark,    J., and Stenman, G. (2004). Altered Notch signaling resulting from    expression of a WAMTPI-MAML2 gene fusion in mucoepidermoid    carcinomas and benign Warthin's tumors. Exp Cell Res 292, 21-28.-   Filler, G., Neuschulz, 1., Vollmer, I., Amendt, P., and Hocher, B.    (2000). Tacrolimus reversibly reduces insulin secretion in    paediatric renal transplant recipients. Nephrol Dial Transplant 15,    867-871.-   Gonzalez, G. A., and Montminy, M. R. (1989). Cyclic AMP stimulates    somatostatin gene transcription by phosphorylation of CREB at    Serine 133. Cell 59, 675-680.-   Goodman, R. H., and Smolik, S. (2000). CBP1p300 in cell growth,    transformation, and development. Genes Dev 14, 1553-1577.-   Hall, R. K. & Granner, D. K. Insulin regulates expression of    metabolic genes through divergent signaling pathways. J Basic Clin    Physiol Pharmacol 10, 119-33 (1999).-   Hanson, R. W. & Reshef, L. Regulation of phosphoenolpyruvate    carboxykinase (GTP) gene expression. Annu Rev Biochem 66,    581-611(1997).-   Herzig, S. et al. CREB Regulates Hepatic Gluconeogenesis via the    Co-activator PGC-1. Nature 413, 179-183 (2001).-   Hogan, P. G., Chen, L., Nardone, J., and Rao, A. (2003).    Transcriptional regulation by calcium, calcincurin, and NFAT. Genes    Dev 17, 2205-2232.-   Horike, N., Takemori, H., Katoh, Y., Doi, J., Min, L., Asano, T.,    Sun, X., Yamamoto, H.,

Kasayama S., Muraoka, M., Nanaka, Y., Okamoto, M.,(2003).Adipose-specific expression, phosphorylation of Ser⁷⁹⁴ in insulinreceptor substrate-1, and activation in diabetic animals of salt-inducedkinase-2. J. Biol. Chem. 278:18440-7.

-   Hui, H., Nourparvar, A., Zhao, X. and Perfetti, R. (2003).    Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting    cells via a cyclic 5′-adenosine monophosphatedependent protein    kinase A- and a phosphatidylinositol3-kinase-dependent pathway.    Endocrinology 144, 1444-1455.-   Iourgenko, V., Zhang, W., Mickanin, C., Daly, I., Jiang, C.,    Hexham, J. M., Orth, A. P., Miraglia, L., Meltzer, J., Garza. D., et    al. (2003). Identification of a family of cAMP response    element-binding protein coactivators by genome-scale functional    analysis in mammalian cells. Proc Natl Acad Sci USA 100, 12 147-52.-   Jhala, U. S., Canettieri, G., Screaton, R. A., Kulkarni, R. N.,    Krajewski, S., Reed, J., Walker, J., Lin, X., White, M. and    Montminy, M. (2003). cAMP promotes pancreatic beta-cell survival via    CREB-mediated induction of LRS2. Genes Dev 17, 1575-1580.-   Kahn, B. B., Alquier, T., Carling, D. & Hardie, D. G. AMP-activated    protein kinase: Ancient energy gauge provides clues to modern    understanding of metabolism. Cell Metabolism 1, 15-25 (2005).-   Kasper, L. H., Boussouar, F., Ney, P. A., Jackson, C. W., Rehg, J.,    van Deursen, J. M., and Brindle, P. K. (2002). A    transcription-factor-binding surface of coactivator p300 is required    for haematopoiesis. Nature 41 9, 738-743.-   Katoh, Y. et al., (2004). Salt-inducible kinase (SIK) isoforms:    their involvement in steroidogenesis and adipogenesis. Mol Cell    Endocrinol 217:109-12.-   Katoh, Y. et al. Salt-inducible kinase-1 represses cAMP response    element-binding protein activity both in the nucleus and in the    cytoplasm. Eur J Biochem 271, 4307-19 (2004).-   Koo, S. H. et al. PGC-1 promotes insulin resistance in liver through    PPAR-alphadependent induction of TRB-3. Nat Med (2004).-   Koo, S. H., et al. The CREB coactivator TORC2 is a key regulator of    fasting glucose metabolism. Nature 437, 1109-1114 (2005).-   Kornhauser, J. M., Cowan, C. W., Shaywitz, A. J., Dolmetsch. R. E.,    Griffith, E. C., Hu, L. S., Haddad, C., Xia, Z., and    Greenberg, M. E. (2002). CREB transcriptional activity in neurons is    regulated by multiple, calcium-specific phosphorylation events.    Neuron 34, 221-233.-   Kwok, R., Lundblad, J., Chrivia, J., Richards, J., Bachinger, H.,    Brennan, R., Roberts, S., Green, M., and Goodman, R. (1994). Nuclear    protein CBP is a coactivator for the transcription factor CREB.    Nature 370, 223-226.-   Lemaigre, F. P., Ace, C. I., and Green, M. R. (1993). The cAMP    response element binding protein, CREB, is a potent inhibitor of    diverse transcriptional activators. Nucleic Acids Res 21, 2907-291    1.-   Lin, J. et al. Defects in adaptive energy metabolism with CNS-linked    hyperactivity in PGC-1 alpha null mice. Cell 119, 12 1-35 (2004).-   Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., Mize, G. J.,    Morris, D. R., Garvik, B. M., and Yates, J. R., 3rd (1999). Direct    analysis of protein complexes using mass spectrometry. Nat    Biotechnol 17, 676-682.-   Lizcano, J. M., Goransson, O., Toth, R., Deak, M., Morrice, N. A.,    Boudeau, J., Hawley, S. A., Udd, L., Makela, T. P., Hardie, D. G.,    and Alessi, D. R. (2004). LKB1 is a master kinase that activates 13    kinases of the AMPK subfamily, including MARWAR-]. Embo J 23,    833-843.-   Lochhead, P. A., Salt, I. P., Walker, K. S., Hardie, D. G. &    Sutherland, C. 5-aminoimidazole-4-carboxamide riboside mimics the    effects of insulin on the expression of the 2 key gluconeogenic    genes PEPCK and glucose-6-phosphatase. Diabetes 49, 896-903 (2000).-   MacCoss, M. J., McDonald, W. H., Saraf, A., Sadygov, R., Clark, J.    M., Tasto, J. J., Gould, K. L., Wolters, D., Washbum, M., Weiss, A.,    et al. (2002). Shotgun identification of protein modifications from    protein complexes and lens tissue. Proc Natl Acad Sci USA 99,    7900-7905.-   Mayr, B., and Montminy, M. (2001). Transcriptional Regulation by the    Phosphorylation Dependent Factor CREB. Nature Reviews-Molecular Cell    Biology 2, 599-609.-   Newgard, C. B., and McGarry, J. D. (1995). Metabolic coupling    factors in pancreatic beta-cell signal hansduction. Annu Rev Biochem    64, 689-719.-   Newman, J. R., and Keating, A. E. (2003). Comprehensive    identification of human bZIP interactions with coiled-coil arrays.    Science 300, 2097-210 I.-   Okamoto, M., Takemori, H., and Katoh, Y. (2004). Salt-inducible    kinase in steroidogenesis and adipogenesis. Trends Endocrinol Metab    15, 21-26.

Okamura, H., Arambum, J., Garcia-Rodriguez, C., Viola, J. P., Raghavan,A, Tahiliani, M., Zhang, X., Qin, J., Hogan, P. G., and Rao, A. (2000).Concerted dephosphorylation of the transcription factor NFAT1 induces aconformational switch that regulates transcriptional activity. Mol Cell6, 539-550.

-   Parker, D., Jhala, U., Radhakrishnan, I., Yaffe, M., Reyes, C.,    Shulman, A., Cantley, L., Wright, P., and Montminy, M. (1998).    Analysis of an Activator:Coactivator Complex Reveals an Essential    Role for Secondary Structure in Transcriptional Activation.    Molecular Cell 2, 353-359.

Radhakrishnan, L, G. C. Perez-Alvarado, Parker, D., Dyson, H. J.,Montminy, M., and Wright, P. E. (1997). Solution structure of the KIXdomain of CBP bound to the transactivation domain of CREB: a model foractivator-coactivator interactions. Cell 91, 741-752.

-   Radziuk, J., Bailey, C. J., Wiernsperger, N. F. & Yudkin, J. S.    Metformin and its liver targets in the treatment of type 2 diabetes.    Curr Drug Targets Immune Endocr Metabol Disord 3, 151-69 (2003).-   Sakamoto, K., Goransson, O., Handie, D. G. & Alessi, D. R. Activity    of LKB 1 and AMIPK-related kinases in skeletal muscle: effects of    contraction, phenformin, and AICAR. Am J Physiol Endocrinol Metab    287, E310-7 (2004).-   Saltiel, A. & Kahn, C. R. Insulin signalling and the regulation of    glucose and lipid metabolism. Nature 414, 799-806 (2001).-   Sasaki, K. et al. Multihormonal regulation of phosphoenolpyruvate    carboxykinase gene transcription. J Biol Chem 259, 15242-15251    (1984).-   Schwaninger, M., Blume, R., Kruger, M., Lux, G., Oetjen, E., and    Knepel, W. (1995). Involvement of the Ca(2+)-dependent phosphates    calcineurin in gene transcription that is stimulated by cAMP through    cAMP response elements. J Biol Chem 270, 8860-8866.-   Screaton, R. A. et al. The CREB coactivator TORC2 functions as a    calcium- and cAMP-sensitive coincidence detector. Cell 119, 61-74    (2004).-   Shaw, R. J., Kosmatka, M., Bardeesy, N., Hurley, R. L., Witters, L.    A., DePinho, R. A., and Cantley, L. C. (2004). The tumor suppressor    LKB1 kinase directly activates AMP activated kinase and regulates    apoptosis in response to energy stress. Proc Natl Acad Sci USA 101,    3329-3335.-   Shaywitz, A. J., and Greenberg, M. E. (1999). CREB: A    Stimulus-Induced Transcription Factor Activated by A Diverse Array    of Extracellular Signals. Annu Rev Biochem 68, 821-861.-   Sheng, M., Thompson, M. A., and Greenberg, M. E. (1991). CREB: A    Ca-Regulated Transcription Factor Phosphorylated by    Calmodulin-Dependent Kinases. Science 252, 1427-1430.-   Tabb, D. L., MacDonald, W. H., and Yates, J. R. (2002). DTASelect    and Contrast: tools for assembling and comparing protein    identifications from shotgun proteomics. Journal of Proteome    Research 1, 21-26.-   Tonon, G., Modi, S., Wu, L., Kubo, A., Coxon, A. B., Komiya, T.,    O'Neil, K., Stover, K., El-Naggar, A., Griffin, J. D., et al.    (2003). t(11;19)(q21;p13) translocation in mucoepidermoid carcinoma    creates a novel fusion product that disrupts a Notch signaling    pathway. Nat Genet 33, 208-213.-   Voon, J. et al. Control of hepatic gluconcogenesis through the    transcriptional coactivator PGC-1. Nature 413, 13 1-138 (2001).-   Woods, A, Johnstone, S. R., Dickerson, K., Leiper, F. C., Fryer, L.    G., Neumann, D., Schlattner, U., Wallimann, T., Carlson, M., and    Carling, D. (2003). LKB1 is the upstream kinase in the AMP-activated    protein kinase cascade. Curr Biol 13, 2004-2008.-   Yamauchi, T. et al. Adiponectin stimulates glucose utilization and    fatty-acid oxidation by activating AMP-activated protein kinase. Nat    Med 8, 1288-95 (2002).

1. A method of screening test compounds to determine if such compoundsare capable of enhancing islet cell activity and/or survival, saidmethod comprising determining the effect of test compounds on one ormore of: the transport of a Transducer Of Regulated CREB (TORC) from thecytoplasm into the nucleus of an islet cell, the interaction between aTransducer Of Regulated CREB (TORC) and a member of the 14-3-3 family ofproteins, or the level of phosphorylation of a Transducer Of RegulatedCREB (TORC), wherein one or more of the following, in the presence oftest compound, is indicative of a compound which is capable of enhancingislet cell activity and/or survival: enhanced transport of TORC from thecytoplasm into the nucleus of an islet cell, disruption of theinteraction between TORC and a member of the 14-3-3 family of proteins,or a reduction in the level of phosphorylation of TORC.
 2. The method ofclaim 1 wherein said TORC is TORC1, TORC2 or TORC3.
 3. The method ofclaim 1 wherein phosphorylation of TORC occurs at a position comparableto Ser171 of TORC2.
 4. A method of screening test compounds to determineif such compounds are capable of promoting CREB-mediated gene expressionin islet cells, said method comprising determining the effect of testcompounds on one or more of: the transport of a Transducer Of RegulatedCREB (TORC) from the cytoplasm into the nucleus of an islet cell theinteraction between a Transducer Of Regulated CREB (TORC) and a memberof the 14-3-3 family of proteins, or the level of phosphorylation of aTransducer Of Regulated CREB (TORC), wherein one or more of thefollowing, in the presence of test compound, is indicative of a compoundwhich is capable of promoting CREB-mediated gene expression in isletcells: enhanced transport of TORC from the cytoplasm into the nucleus ofan islet cell, disruption of the interaction between TORC and a memberof the 14-3-3 family of proteins, or a reduction in the level ofphosphorylation of TORC.
 5. A method of screening test compounds todetermine whether such compounds affect transport of a Transducer OfRegulated CREB (TORC) from the cytoplasm into the nucleus of a cell,said method comprising determining the effect of test compounds on thetransport of TORC from the cytoplasm into the nucleus of said cell,wherein said cell is selected from the group consisting of an isletcell, a muscle cell, a liver cell, and an adipose cell.
 6. The method ofclaim 5 wherein said test compound enhances the transport of TORC fromthe cytoplasm into the nucleus of said cell.
 7. The method of claim 6wherein said cell is an islet cell.
 8. The method of claim 5 whereinsaid test compound reduces the transport of TORC from the cytoplasm intothe nucleus of said cell.
 9. A method of screening test compounds todetermine whether such compounds affect the interaction between aTransducer Of Regulated CREB (TORC) and a member of the 14-3-3 family ofproteins, said method comprising determining the effect of testcompounds on the interaction between TORC and a member of the 14-3-3family of proteins.
 10. The method of claim 9 wherein said test compoundenhances the interaction between TORC and a member of the 14-3-3 familyof proteins.
 11. The method of claim 9 wherein said test compounddisrupts the interaction between TORC and a member of the 14-3-3 familyof proteins.